In water environment, nitrogen (N) and phosphorus (P) are biochemically dependent nutrients following the co-limitation concept for algae growth under mixotrophic mode. From a practical viewpoint, algae growth may not bring about significant change of the background nutrient concentration of an actual waterbody in contrast to a conventional batch system. In order to better understand the growth pattern of microalgae in aquatic environments, a series of experiments were conducted under stably controlled N-P levels for studying the N-P coupling effect on mixotrophic Chlorella vulgaris growth process, with attention paid to the physiological and biochemical characteristics. It was found that within the concentration range of N = 1–8 mg·L−1 and P = 0.1–1.0 mg·L−1, the variation of the N-P level slightly affected the specific growth rate, but significantly influenced nutrients uptake, biomass dry weight, chlorophyll contents of the grown C. vulgaris. The biochemical and elemental composition of the microalgae tended to be more sensitive to the N-P concentrations and ratios in the lower nutrient range (1–2 mg N·L−1, 0.1–0.4 mg P·L−1) in which the highest N and P conversion rates were gained as 90.18 ± 1.23% and 60.47 ± 1.59%, respectively. The P assimilation and conversion efficiencies were much affected by both N and P supplies, while the P supply showed little influence on N assimilation and conversion efficiencies. It was also noticed that the N level greatly affected the metabolic pathway involving nutrient assimilation, carbohydrate fixation and monosaccharide profile, resulting in conversion of the dominant fraction of protein at N ≤ 2 mg·L−1 into other biochemical compositions including lipids at N ≥ 3 mg·L−1. The fatty acid methyl esters (FAMEs) composition tended to differ with varied nutrient levels. These findings may deepen our understanding of algal growth in aquatic environment and provide perspective for eutrophication control.