Intratumoral injection of various effector cells combined with oncolytic adenovirus expressing antitumor cytokines exert an effective antitumor immune effect by oncolysis and altering the tumor microenvironment. However, this combination therapy had certain limitations. When used in high concentrations, effector cells and oncolytic viruses can spread rapidly to surrounding non-target tissues. And because both therapies used in combination are immunogenic and exhibit shorter biological activity, multiple injections were required to attain an adequate therapeutic index. To overcome these drawbacks, we encapsulated gelatin-based hydrogel capable of co-deliver oncolytic adenovirus armed with IL12 and IL15 (CRAd-IL12-IL15) and CIK cells for enhancing and prolonging the antitumor effects of both therapies after a single intratumoral injection. The injectable and biodegradable hydrogel reduced the dispersion of high-dose oncolytic adenovirus and CIK cells from the injection site to the liver and other non-target tissues. In this study, a novel oncolytic adenoviral vector CRAd-IL12-IL15 was constructed to verify the cytokine expression and oncolytic ability, which can upregulate the expression levels of Bcl-2, Cish and Gzmb in tumor cells. The CRAd-IL12-IL15 + CIKs/gelatin treatment maintained sustained release of CRAd-IL12-IL15 and active CIK cells over a longer period of time, attenuating the antiviral immune response against adenovirus. In conclusion, the results suggested that hydrogel-mediated co-delivery of CRAd-IL12-IL15 and CIK cells might be a an approach to overcome limitations. Both treatments could be effectively retained in tumor tissue and sustained to induce potent anti-tumor immune responses with a single administration.