The Home Secretary, Juan Ignacio Zoido, has used Article 155 powers to sack Catalan Police (Mosso) chief Josep Lluis Trapero.
The decision was published in Spain's official gazette (BOE) in the early hours of Saturday morning.
Major Trapero had been appointed to the post on April 11.
He is currently being investigated by the National High Court in Madrid for the crime of sedition, a public order offence in the Spanish Criminal Code that carries a maximum 15-year term.
The investigating judge, Carmen Lamela, ordered Trapero to hand over his passport, not leave the country and appear before a judge every 15 days.
On October 1, the Catalan Police did not act as expected to close some 2,300 polling stations around the region to prevent the illegal vote from taking place.
In the weeks prior to the referendum, there was continued doubt about where his loyalties were and whether or not the Mossos would follow orders from judges or the Spanish Home Office.
In the end, a state-level coordinator, Civil Guard Colonel Diego Pérez de Cobos, was put in charge of all policing for the referendum.
Two days before the vote, Major Trapero ordered his officers not to use force to police the polling stations, and on the morning of Sunday, October 1, Mossos could be seen standing outside the buildings doing nothing or, in at least one case, helping separatists to move ballot boxes into vans.
Trapero was well regarded for his handling of the police response to the terrorist attacks in August in Barcelona and Cambrils.