Since the season re-started after being halted by the COVID-19 pandemic, Real Betis lost their first game back 2-0 to Sevilla and drew 2-2 with Granada in mid-week.
The final straw proved to be Saturday’s 1-0 defeat by Athletic Bilbao, after Sergio Canales missed a late penalty kick.
Real Betis released a statement in the early hours of Sunday saying Rubi had been relieved of his duties.
It added that sporting director Alexis Trujillo, a former Real Betis player, would take charge of the team for the final eight games of the season.
Defeat by Athletic Bilbao left Real Betis 14th in the standings on 34 points after 30 games.
They are eight points above Real Mallorca who occupy the final spot outside of the relegation zone.
Rubi was named Real Betis coach last summer after guiding Espanyol to seventh last season, succeeding Quique Setien who went on to become the FC Barcelona manager last January.