Previous studies of bladder tissue engineering simply seeded cells onto the surface of the material, which makes the cells lack protection and makes it difficult to face the complex in vivo environment. The gelatin methacryloyl (GelMA) hydrogel possesses outstanding biocompatibility and distinctive photo-crosslinking characteristics and is capable of offering a suitable three-dimensional growth environment for cells. This study explored the optimal concentration of GelMA for encapsulating adipose-derived stem cells (ADSCs) and combined it with bladder acellular matrix (BAM) to create a tissue-engineered bladder patch. Results indicated that 10% GelMA more effectively promoted ADSCs proliferation and spreading compared to 7.5% and 12.5% concentrations, which can offer a better extracellular matrix environment for cells. BAM performed as an excellent substrate with mechanical properties and stitchability similar to natural tissues. Animal experiments demonstrated that the encapsulated ADSCs in GelMA enhanced patch vascularization in vivo and BAM-GelMA-ADSCs tissue-engineered bladder patch can repair large-scale bladder defects in beagles and promote bladder tissue regeneration and functional recovery. This photocrosslinking hydrogel-acellular matrix patch provides a protective semi-controlled environment for ADSCs, supporting the growth and viability of encapsulated cells in vivo, while being easy to suture and preventing leakage, and has significant clinical potential.