The culture conditions of HepG2 cells on the polydimethylsiloxane (PDMS)-glass microfluidic chip were optimized, including the cell attachment surface modification, cell seeding density, medium serum concentration and its replacement frequency. The on-chip HepG2 cells culture was compared with the traditional well plate cell culture. It is found that fibronectin (FN) has better cell-adhesive growth ability than polylysine (PLL) and collagen, 1.0×10⁶～2.0×10⁶/ml is the appropriate cell seeding density on chip, increasing the serum volumn fraction to 20% (V/V) is beneficial to the growth of cells on chip, and the cell culture medium should be replaced every 8～16 hours on chip. After optimization, HepG2 cells on chip show the same proliferation tendency and proliferation rate as HepG2 cells on the conventional well plate.