Messi returned to training this week after a calf problem, with the 32-year-old missing Barca's entire programme of pre-season friendlies because of Copa America commitments and his injury.
Although he got several days of training under his belt and appeared to be in fine form in video footage that came out of the sessions, it was reported Messi felt "discomfort" on Saturday (AEST).
Coach Ernesto Valverde also urged fans to be patient in his pre-match news conference a day later, adamant Messi will not be risked if he is anything other than 100 per cent ready to feature.
As such, Messi has been left out of the squad for the visit of Betis, which left Camp Nou with a stunning 4-3 win last season.
There is a place in the selection for Ansu Fati, a 16-year-old winger who impressed in the UEFA Youth League in 2018-2019.
Barca will be aiming to bounce back from a 1-0 opening-day defeat at Athletic Bilbao.