Chlorella vulgaris is a natural feed that plays an important role in cultivation due to its ease of cultivation and high nutritional value. However, its production is hindered by the high cost of the culture media, which makes the overall production expensive. One potential solution is to use a medium made from fermented mother liquor (FML) enriched with NaNO₃, as its nitrogen-phosphorus (N/P) content is beneficial for the growth of C. vulgaris. This study aimed to assess the impact of FML and NaNO₃ substitution as a culture medium for C. vulgaris and to determine the optimal dose. The treatments used in this study were P1 (Walne, serving as the control with an N/P ratio of 17:1), P2 (10:1), P3 (15:1), P4 (17:1), and P5 (20:1). The results demonstrated that media with FML substitution enriched with NaNO₃ can not only replace Walne fertilizer but also outperform it. The optimal dose of FML substitution enriched with NaNO₃ for maximizing chlorophyll-a, chlorophyll-b, and carotenoid content in C. vulgaris on a laboratory scale was found to be the N/P ratio of 15:1, with a population density of 10.92 x 10⁶ cells/ml (day 7), a growth rate of 25 x 10⁶ cells/ml (day 3), chlorophyll-a content of 0.49μg/ ml, chlorophyll-b of 0.46μg/ ml (day 7) and carotenoids of 0.16μg/ ml (day 7).