Biogas utilization through biotechnology represents a potential and novel technology. We propose the microalgal mixotrophic cultivation to convert biogas to microalgae-based biodiesel, in which methanotroph was co-cultured to convert CH4 to organic intermediate (and CO2) for microalgal mixotrophic growth. This study constructed a co-culture of Methylocystis bryophila (methanotroph) and Scenedesmus obliquus (microalgae) with biogas feeding. Compared with the single culture of S. obliquus, higher microalgal biomass but a lower chlorophyll concentration was observed. The organic metabolism-related genes were upregulated, verifying microalgal mixotrophic growth. The stoichiometric calculation of M. bryophila culture shows that M. bryophila tends to release organic matter rather than grow under a low O2 content. M. bryophila rarely grew under five different light intensities, indicating that M. bryophila acts as a biocatalyst in the co-culture. The organic intermediate released by methanotroph increased the maximum biomass of microalgal culture, accelerated nitrogen absorption, accumulated more monounsaturated fatty acids, and improved the adaptation to light. The co-culture of microalgae and methanotroph may provide new opportunities for microalgae-based biodiesel production using biogas as a substrate.