Higher biomass and lipid yields in cells were acquired by co-substrates culture. The synthesis of C18:3n3 in cells was markedly reduced by co-substrates culture. The synthesis of C18:1n9c in cells was stimulated by co-substrates culture. Higher carbohydrate content in cells was induced by co-substrates culture. The biofuel property of C. sorokiniana was enhanced by co-substrates culture.