Abstract: A newly isolated spontaneously flocculating microalgae Parachlorella kessleri F01 was used as the material, and the algae sample in autotrophic cultivation of one-step culture method was used as the control group. A two-step culture method was designed in which glucose was supplemented for mixotrophic cultivation at stage Ⅰ, and nutritional limitation treatments were done at stage Ⅱ, and we studied their effects on the lipid accumulation and flocculation performance of the algae. The algae cell number was determined by a hemocytometer. Biomass was measured via dry weight, and the total lipid was quantified with staining method. The extracellular polymeric substances (EPS) components of algae were analyzed by three-dimensional fluorescence spectroscopy. The results showed that: (1) The optimum glucose concentration was 10 g/L at stage Ⅰ. The alga lipid productivity reached to 204.25 mg/L/d on the 10th harvesting day, which was 16.20 times of that in the control group, and the self-flocculation rate of alga cell was 96.1% after sitting for 12h, which has no significant difference with that in the control group. (2) On the basis of stage Ⅰ, alga cells were cultured for 1 day in stage Ⅱ with different elemental treatments. The alga lipid productivity were 242.64 mg/L/d and 227.61 mg/L/d in the low-glucose group and the low-glucose coupled with low-nitrogen group, respectively, increasing by 18.8% and 11.4% of those at stage Ⅰ. After 4 days cultivation, the alga lipid productions of the four treatment groups including low-glucose, no-glucose, low-nitrogen and the low-glucose coupled with low-nitrogen significantly increased compared with those in the control group and stage Ⅰ. The highest production reached to 3.08 g/L in the low-glucose and low-nitrogen co-treatment group, which was 23.69 times of that in the control group, and increased by 51.0% compared with that in stage Ⅰ. The self-flocculation rates in four treatment groups at stage Ⅱ were nearly above 85.0%, which could satisfy the harvesting requirements. (3) The content of protein tryptophan in alga EPS was positively related to the self-flocculation rate. Different culture treatments could change the protein tryptophan content in EPS of algae cells, and thus affect their flocculation performance. Self-flocculation Parachlorella kessleri F01 is an excellent potential alga for biodiesel production. The two-step culture method can greatly enhance its lipid production. The advantage of self-flocculation combined with two-step culture method is expected to be the key breakthrough to solve the bottleneck of biodiesel production technology of microalgae.