Abstract: For promoting biomass and lipid production in Monoraphidium sp.QLY-1, this study investigated that the effects of glycine betaine ( GB) on the growth and lipid accumulation in QLY-1 in combination with a two-step strategy, which was called heterotrophic and photoinduction cultivation. Results showed that the biomass concentration of heterotrophic and autotrophic algal cells were ( 5.54 ± 0.22) and ( 0.878 ± 0.12) g/L, respectively, and the heterotrophic cells was 5.3 folds higher than that of the autotrophic.Moreover, under high light stress, the lipid content ( 47.37% ± 2.93%) was enhanced by 0.29 folds ( p < 0.05) treated with 5 mmol/L glycine betaine than that of control ( 36.68% ± 1.34%) . Further studies showed that the neutral lipid content was increased to 12.83% ± 0.75% than that of the control, and was no significant influences on the fatty acids composition under 5 mmol/L glycine betaine induction. The results indicated that glycine betaine could significantly increase the production of lipid in microalgae, combination of glycine betaine and two-step cultivation could act as another strategy for promoting the lipid accumulation in microalgae.