Microalgae biomass production with starch wastewater (SW) is a promising approach to realize waste recovery and cost reduction due to the inherent copious nutrients and nontoxic compounds in SW. However, the application of this technique is significantly hindered by low biomass production on account of the poor photosynthetic efficiency of microalgae. In this regard, we proposed a photo-regulation strategy characterized by the adjusting of numbers of light/dark (L/D) cycles, and compositions of light wavelength, which was proved to be an effective method for stimulating intracellular photo electron transfer and enhancing photosynthetic efficiency, to boost microalgae biomass accumulation. Additionally, responses of the microalgae photo-biochemical conversion, and the wastewater treatment performance at various number of L/D cycles and light wavelengths were discussed. The experimental results indicated that the biomass production increased when the L/D period was increased from 2 h:2 h–12 h:12 h. When the L/D period was 2 h:2 h, the biomass production reached a maximum value of 1.28 g L−1, which was 19.6% higher than that of the control group when the L/D period was 12 h:12 h. Furthermore, with respect to microalgae growth under monochromatic light, the maximum biomass concentration (1.25 g L−1) and lipid content (32.2%) of Chlorella were achieved under blue light; whereas, the minimum values were attained under red light (1.05 g L−1 and 19.3%, respectively). When the red light and blue light were mixed and supplied, the microalgae biomass productivity was higher than that under white light, and the highest lipid productivity was 109.0 mg−1 L−1 d under a blue: red ratio of 2:1. Moreover, gas chromatography analysis demonstrated that the methyl in the range of C16–C18 in the system was higher than 70%. Fatty acid methyl esters (FAMEs) containing palmitic acid (C16:0) and oleic acid (C18:1) are beneficial for production of biodiesel, and the quality of fatty acid methyl ester used in biodiesel production can be improved using microalgae cultured under the mixed wavelengths of blue and red. Finally, Chlorella was cultured in PBR and reached the peak concentration of 2.45 g L−1 by semi-continuous process with the HRT regulation.