The heterogeneity of bacterial cells in biofilms is an important property. We performed single-cell sequencing to profile heterogeneous cell populations within Vibrio alginolyticus biofilm. Our analysis has revealed the key functional compartments for the composition and development of biofilms Overall design: After 60 hours of incubation at 28°C on LB agar, Vibrio algalinolyticus developed mature biofilms, which were collected for single-cell sequencing to investigate the functional heterogeneity of bacterial cells within the biofilm.