The BBC announced on Wednesday that the play will broadcast its final show in March 2022.
The company said it had been"incredibly proud" of this series but it"needed to make tough decisions to make space for new opportunities".
Approximately 250 people working on the series - like actors, camera operators, hair and engineers along with make-up professionals - will probably be impacted by the choice.
Holby City won a Bafta for best continuing drama in 2008 and regularly attracts over 3 million viewers.
"We're extremely proud of Holby City but it's with fantastic sadness that we're declaring that after 23 decades, the series will finish on screen in March of next year," the BBC said in a statement.
"We sometimes need to make difficult choices to make space for new opportunities and as an element of the BBC's commitment to create more programmes across the united kingdom, we've taken the challenging decision to bring the series to a close so as to reshape the BBC's play slate to reflect, reflect and serve all areas of the nation."
It added:"We want to take this chance to thank the wonderful staff at BBC Studios and the cast and crew who've been involved from the series since 1999.
"Holby was a stalwart with viewers, delighting millions of viewers per week and winning countless awards with a persuasive mixture of cutting edge medical stories and volatile private stories.
"We anticipate working with the staff within the coming months to make sure that if it finishes, Holby goes out on a top."
The two Holby City, and Casualty are located in the fictional county of Wyvern, South West England.
However, the Majority of the on-set filming for Holby City occurs in the BBC's Elstree Centre in Hertfordshire, in Addition to shots from around Bristol.
In March, the BBC announced its strategy to move a number of its key departments and employees around the nation so as to make the company more reflective of the UK as a whole.
"Our mission has to be to provide for the whole of the united kingdom and make sure every family will get value from the BBC," said director general Tim Davie.
"These programs will get us nearer to viewers, create investment and jobs, and create and cultivate new talent"
Racking up over 1,000 episodes over 23 show, Holby City has also been shown to be a fertile breeding ground for several top acting ability over time.
Killing Eve celebrity Jodie Comer received among her very first acting jobs to the programme, as did fellow Bafta-winner and Dark Panther celebrity Letitia Wright, Peaky Blinders celebrity Joe Cole and Lee Ryan in the boyband Blue.
Already-established celebrities like Adrian Edmondson and Patsy Kensit were given components, although the likes of Maureen Lipman, chat show host Paul O'Grady and comic Romesh Ranganathan also have appeared as guests.
The series has held its own place at the BBC One program on a Tuesday night for several decades, tackling several topical problems on the way.
Among the very memorable storylines involved the separation of the conjoined Tan twins, in 2008. The operation was completed from the series's longest-serving personality Dr Ric Griffin, played by Hugh Quarshie.
A couple of years before, it revealed Professor Elliot Hope's wife Gina travelling to Switzerland to end her life after fighting with motor neurone disease.
A number of the hospital fictional employees have themselves also confronted health problems. In 2014 youthful junior physician Zosia March was diagnosed with bipolar illness, after enduring a breakdown after her mother's departure. Dr Arthur Digby has been diagnosed with melanoma a couple of decades later.
The hospital came under siege during a shooting spree at a 2017 episode.
The next year, a unique show entitled The Christmas Waltz aided to mark the 70th anniversary of the National Health Service. It watched characters describing what the NHS means to these as they tried to obtain an outbreak of norovirus in check.