Human embryonic kidney cells are the host of adenovirus type-5 (Ad5) amplification. An Ad5-vector-based COVID-19 vaccine has been proven to be tolerated and immunogenic in healthy adults. Therefore, a rationally designed scaffold for culturing human embryonic kidney cells is useful for further studying its mechanism of action. Herein, a three-dimensional layered reticulated polycaprolactone (PCL) scaffold coated with poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) was developed to proliferate human embryonic kidney cells and to be used to amplify the Ad5 vector. The results indicate that PCEC improves the hydrophilicity and the cell culture ability of PCL cell culture scaffolds, resulting in a three times higher cell proliferation ratio of human embryonic kidney cells compared with those grown on bare PCL cell culture scaffolds. Meanwhile, the cytotoxicity test results showed that the scaffold material is noncytotoxic. This work provides an effective and scalable method for the in-depth study of adenoviruses.