Suppression of Tumorigenicity 2 (ST2) is a member of the IL-1 receptor family, which includes transmembrane (ST2L) and soluble (sST2) isoforms. sST2 functions as a decoy receptor for Interleukin-33 (IL-33), thereby blocking the activation of the IL-33/ST2L signaling axis, which is essential for tissue repair and immune regulation. Clinical evidence indicates that elevated sST2 levels are associated with increased disease severity in conditions such as ulcerative colitis (UC), cardiovascular disease, and asthma. However, current antibodies cannot reliably distinguish between sST2 and its membrane-bound isoform ST2L, limiting their effectiveness for diagnostic and therapeutic use. To address this limitation, we developed an antibody that selectively targets sST2. Using a phage display library, we identified a single-chain variable fragment (scFv) with high specificity for a unique five amino acid sequence (SKECF) located at the C-terminus of sST2. Our parental scFv showed high selectivity for sST2 with minimal cross-reactivity to ST2L, as demonstrated by both flow cytometry and immunoprecipitation. Molecular simulations identified key binding residues, allowing the design of four scFv mutants, three of which displayed improved binding in surface plasmon resonance (SPR) analyses. The A183YL2 mutant exhibited a 3.4-fold increase in binding affinity, while G100WH3 demonstrated reduced binding due to unfavorable conformations. This study presents an anti-sST2 scFv with enhanced specificity and affinity, offering a promising tool for the diagnosis and treatment of inflammatory diseases, in which sST2 interferes with IL-33-mediated tissue repair.