In this paper, the effects of sodium selenite on the growth of marine cultured Chlorella pyrenoidosa and the transformation ability of organic selenium, as well as the main selenium form in the selenoprotein-rich Chlorella were studied. The optimal selenium-enriched culture concentration was determined by adding 2 μg/mL to 50 μg/mL sodium selenite in batches, and the content of three main selenium forms in algae was determined by HPLC: sodium selenite, selenocysteine and selenomethionine. The results showed that the concentration of sodium selenite should not be too high, otherwise the growth of Chlorella pyrenoidosa was inhibited. When the concentration of selenium was 2 μg/mL, the biomass of Chlorella was high, and the content of organic selenium reached 301.40 μg/g, accounting for 83.24% of the total selenium content. HPLC analysis showed that the main forms of organic selenium in Selenium-enriched Chlorella were selenocysteine and selenomethionine under the culture conditions, and the unconverted sodium selenite accounted for only 23.02% of the three major selenium forms determined. The above results indicate that under seawater culture, the suitable selenium concentration for selenium-enriched culture of Chlorella is 2 μg/mL. Under these conditions, the Chlorella pyrenoidosa grows well; the inorganic selenium is effectively converted, and the content of organic selenium is high, which can meet the requirement of selenium enrichment.