This invention discloses a production process for recombinant human endostatin adenovirus in order to optimize the procedure for small batch and mass industrialization. Exemplary process include steps of: (1) fermentation of eukaryotic cells (HEK293 cells) in the condition of 37° C. and 5% CO2; (2) adenovirus infection; (3) collection of diseased cells; (4) freezing and thawing; (5) concentration by ultrafiltration; and (6) preparation and packaging of recombinant human endostatin adenovirus. The process is controllable and easy to operate. The concentration of adenovirus titers can reach 1.0×1012−3.0×1012 vp/ml.