In rice (Oryza sativa L.), the short-chain dehydrogenase protein OsABA2 plays a crucial role in regulating abscisic acid (ABA) biosynthesis. However, little is known about the other proteins that interact with OsABA2 to regulate ABA biosynthesis. Using yeast two-hybrid screening, we identified a novel OsABA2 interacting protein OsPP2C55, which contains a serine/threonine phosphatase (family 2C) catalytic domain. The yeast two-hybrid (Y2H) assay and firefly luciferase complementary imaging (LCI) assay confirmed these interactions. Subsequent studies revealed that saline–alkaline stress significantly downregulated OsPP2C55 gene expression. Meanwhile, we constructed ospp2c55 CRISPR gene knockout (ospp2c55-KO) plants using Agrobacterium genetic transformation. Compared with wild-type plants, ospp2c55-KO plants under saline–alkaline stress exhibited significantly elevated OsABA2 protein levels, leading to substantial increases in ABA content. In addition, ospp2c55-KO plants demonstrated heightened sensitivity to ABA during seed germination. Moreover, ospp2c55-KO plants improved the survival rate and stress-related indices of rice seedlings under saline–alkaline stress, and upregulated the expression of genes related to adversity stress (OsNCED1, OsNCED3, OsABA2, OsSODCc2, and OsCatB). We found that OsPP2C55 plays a negative regulatory role in ABA biosynthesis and saline–alkaline stress tolerance in rice.