Lymphoid-specific tyrosine phosphatase (Lyp), a critical signaling regulator of immune cells, is associated with various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Recent research suggests that Lyp is a potential drug target for autoimmune diseases. Herein, we applied a target-ligand interaction-based virtual screening method to identify novel Lyp inhibitors. Nine Lyp inhibitors with novel scaffolds were identified with eight reversible inhibitors (Ki values ranged from 2.87 to 28.03 μM) and one covalent inhibitor (Ki = 40.98 ± 13.19 μM). The top four compounds (A2, A15, A19, and A26) displayed selectivity over other phosphatases in preliminary experiments, and kinetic analysis indicated that these compounds are competitive inhibitors of Lyp. Compounds A15 and A19 up-regulated TCR (T cell receptor) mediated signaling and transcriptional activation through inhibition of Lyp activity in T cells. The new chemotypes of Lyp selective inhibitors identified through the target-ligand interaction-based virtual screening may provide new leads for Lyp targeted therapeutic development.