<p>The low survival rate and compromised functionality of β cells present significant obstacles in islet transplantation for diabetes management. Recent studies indicate that the sensitivity of β cells to their microenvironment may be a contributing factor for these. In this study, Biotin-^DFYIGSRGD hydrogel (Supragel) was employed to replicate the microenvironment of mouse β cells (MIN6 cells) and human islets, for enhancing cell viability, functionality, and structural integrity. Compared with conventional two-dimensional cell culture methods utilizing cell plates, in the present method, MIN6 cells could adhere to the gel and self-organize into cell spheroids, which resulted in increased synthesis of insulin and glucagon in MIN6 cells. Furthermore, the hydrogel effectively preserved the integrity of human islets and extended their <i>in vitro</i> culture period. This improvement may be attributed to the optimal hardness of the gel, which facilitated cell adhesion and promoted vascularization. Additionally, the gel exhibited sustained <i>in vivo</i> retention over 120 days, undergoing gradual degradation without eliciting significant inflammatory responses. These properties establish it as a promising encapsulation material for pancreatic islet cells or organoids, supporting long-term cell survival, function, and integration with host tissues. The study highlights the potential of Biotin-^DFYIGSRGD hydrogel as an islet culture platform for diabetes treatment.</p>