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This study compared the growth and biomass production of Isochrysis galbana under hetero-, mixo-, and phototrophic conditions using different organic carbon sources. The growth of I. galbana was inhibited in heterotrophy but was enhanced in mixotrophy compared to that in phototrophy. Subsequently, the influences of organic carbon and environmental factors (light and salinity) on the growth of I. galbana were further investigated. Algal dry weight increased as glycerol concentrations increased from 0 to 200 mmol and the highest algal production occurred at 50 mmol glycerol. At a range of light intensities of 25–200 μmol photons m−2 s−2, the highest algal growth rate occurred at 100 photons μmol m−2 s−2. The growth of I. galbana was significantly affected by photoperiod, and the maximal dry weight was obtained at 12 h light and 12 h dark. In the salinity test, I. galbana could grow in a wide range of salinities from 10 to 65‰, but the 35‰  salinity was optimal. This study suggests that the growth and production of I. galbana can be improved using mixotrophic culture at 50 mmol glycerol in 35‰  salinity.