Porcine urinary bladder is one of the most used organs to fabricate extracellular matrix (ECM) hydrogel. Although there are two different ECM types inside a bladder, i.e., urinary bladder matrix (UBM) and a subtype ECM (sECM), most studies have only employed UBM for hydrogel fabrication, and overlooked the potential use of sECM. In another aspect, the delamination of UBM from bladders is a time-consuming process; consequently, the use of the whole bladder (WB) will likely increase production yield. Therefore, the objective of this study was to fabricate hydrogels from sECM and WB and compare them to UBM. The results indicated that different layers of the bladder shared almost the same biochemical composition. In terms of gelation kinetics, rheology and morphology, although hydrogels from UBM and sECM exhibited some discrepancies, those from UBM and WB interestingly possessed almost the same characteristics. In in vitro studies, all the hydrogels possessed nearly the same biochemical effects towards L929 viability and C2C12 differentiation. These results could preliminarily indicate that the use of sECM should no longer be ignored, and WB could be a promising substitution for UBM hydrogels, eliminating the need for time-consuming delamination processes, as well as increasing the possibility of mass production.