Owing to the complexity of the lipid composition of microalgae and the large difference in polarity, a method to determine its multiple functional lipids without relying on mass spectrometry remains lacking. To tackle this challenge, this study developed a green and eco-friendly supercritical fluid chromatography-evaporative light scattering detection (ELSD) method. The separation was conducted using a Diol column through gradient elution with a mobile phase consisting of supercritical carbon dioxide-methanol. The flow rate was 0.5 mL•min-1. Detection was performed using an ELSD, and the compounds were quantified using an external standard method. The results indicated that this method could separate and determine eight functional lipids within 35 minutes. Compared with traditional liquid phase methods, which require approximately 40 mL of organic reagents, our method requires only 12.28 mL. Good linearity was obtained within the range of 0.01~1.0 mg•mL-1 (R2 ≥ 0.990 2), with limits of detection ≤ 0.74 μg•mL-1, relative standard deviation ≤ 1.19%, and spiked recovery rates ranging from 92.12% to 105.78%. Significant differences were observed in the contents of functional lipids in six different microalgae samples. Specifically, Chlorella vulgaris and Nannochloropsis sp. demonstrated higher contents of glycerides (approx. 53.08 and 57.91 mg•g-1, respectively), glycerolipids, (approximately 39.90 and 32.76 mg•g-1, respectively), and phospholipids (approx. 19.40 and 29.54 mg•g-1, respectively) than the other samples, thus exhibiting potential for serving as dietary supplement sources of glycerolipids and phospholipids The established method is highly sensitive, accurate, and environmentally friendly and enables the separation and quantification of lipids spanning a wide polarity range, from low- to high-polarity, within a single injection. Furthermore, the method fulfills the need for rapid and precise lipid analysis of microalgae samples and is applicable for lipid profiling in diverse food matrices containing specific lipid components.