Regulating stem cells by microRNA (miRNA) is promising in regenerative medicine. However, non-viral transfection is usually transient while stably lentiviral transfection is accompanied with oncogenic risk. In the study, we explored the feasibility to retain the microRNAs within biopolymer hydrogels for their long-lasting working and sustaining stimulation of target gene. miRNA-21 (MiR-21), a reported microRNA enhancing the therapeutic potential of mesenchymal stem cells (MSCs) was used. We demonstrated that miR-21 could be efficiently retained within collagen hydrogel after forming complex with cationic polymer polyethylenimine (PEI). Due to the electronic interaction with positively charged PEI, the release of miR-21 was largely prevented during 2week incubation (<20%), while free miR-21 encapsulated in hydrogels was largely released (>50%). When MSCs were cultivated in the PEI/miR-21-incorporated hydrogels, the sustained activation of targeted gene HIF-1 was observed, resulting in the sustaining up-regulation of several downstream therapeutic cytokines. Then, the hydrogels encapsulating miR-21/PEI were coated onto tissue plate for MSC cultivation, which further confirmed the long-lasting retention and efficacy of miR-21 on the plate surface. In addition, under H2O2-simulated stress condition, we also demonstrated that the anti-apoptotic capacity of MSCs was significantly improved when growing on miR-21-retained hydrogels. Our study provided a safe and promising method for long-lasting stem cell regulation with miRNAs.