Heterogeneous catalysts with excellent activity and stability are challenges for biodiesel production. Herein, two typical biomass derived mesoporous activated carbon (by chemical activation (CAC) and physical activation (PAC)) supported nano-CaO catalysts were prepared by regulated methods. By simple impregnation of calcium nitrate, the catalytic biodiesel yield over CaO/CAC was distinctly increased from 9.16% to 98.4% with the extreme elimination of phosphate groups in the carbon matrix. The catalytic activity of CaO/PAC was enhanced from 70.1% to 96.0% with a deposition-precipitation method for nano-CaO introduction. The abundant surface oxygenated groups of CAC could dispersively anchor the calcium species, leading to dispersion of stable nano-CaO (∼11.7 nm) in the mesopores, high basic sites concentration and excellent catalytic activity and stability. The developed mesopores with large diameter promote disperse growth of calcium oxide, diffusion rate and tolerance of bulky reaction molecules, and accessibility to the active sites, resulting in the improvement of catalytic performance. Both the phosphate groups elimination and deposition-precipitation technique effectively improved the surface area, mesopore volume, basic sites density and biodiesel yield of supported nano-CaO catalysts. The optimal CAC enhanced the catalytic activity 16-fold compared to pristine CaO, and CaO/CAC catalyst presented excellent reusability for transesterification without post-treatment.