Pancreatic cancer organoids (PCOs) have gained extensive attention as promising in vitro models that can advance our understanding of translational cancer biology and biomedical research. To date, PCOs are mostly cultured in animal-derived matrices, which are limited by their low similarity with native tumors due to batch-to-batch variations, stringent operating conditions, and uncontrollable physicochemical properties. Here, we developed a more controllable hydrogel matrix comprising sodium alginate (NaA) and hyaluronic acid (HA) that can mimic the mechanical properties of native tumor tissue, such as extracellular matrix (ECM) components and stiffness. The PCOs cultured in the hydrogel matrix exhibited similar viability and growth rate with that in commercial Matrigel. Furthermore, we observed improvements of PCOs in 1% NaA-HA hydrogel matrices over tumor-specific features observed previously in animal-derived matrices. Transcriptional analysis revealed the activation of signaling pathways associated with ECM organization in the PCOs generated in hydrogel. Moreover, we noted that the biomimetic stiffness of hydrogel enhanced the drug resistance of PCOs of conventional chemotherapy agents but improved the sensitivity to targeted antitumor drugs (Erlotinib) of the PCOs with EGFR mutation. This work represents foundation for the customizing hydrogel stiffness that can be utilized to mimic the native tumor tissue, as well as a new platform for performing pancreatic cancer research and antitumor drug screening in the future.