In recent years, the usage of nitroaniline has played a vital role in pharmaceutical formulations as it is a crucial ingredient in the synthesis of pesticides and dyes. However, the level of nitroaniline existing in industrial waste keeps rising the environmental contamination. Thus, monitoring of active nitro-residuals becomes more significant in reducing the toxicity of the ecosystem. Therefore, we have taken an attempt to evaluate the hazardous pollutant 2-nitroaniline (2-NA) using the electrocatalyst viz., tin-doped bismuth oxide inserted on a biopolymer silk fibroin composite modified glassy carbon electrode (Sn–Bi₂O₃/SF@GCE). The Sn–Bi₂O₃/SF nanocomposite was synthesized through hydrothermal and co-precipitation methods. The physicochemical properties of the prepared Sn–Bi₂O₃/SF hybrid composite were examined by conventional microscopy and spectroscopic techniques like FE-SEM, HR-TEM, XRD, FTIR, Raman, and XPS. Furthermore, the bio-mineralized Sn–Bi₂O₃/SF@GCE displayed a wide linear range (0.009 μM–785.7 μM) and a lower detection limit (3.5 nM) with good sensitivity for 2-NA detection under the optimum conditions. The result shows that the Sn–Bi₂O₃/SF-modified GCE has good reproducibility, repeatability, and excellent selectivity for 2-NA detection in the presence of other co-interfering compounds. Moreover, the practical applicability of Sn–Bi₂O₃/SF@GCE sensors was investigated for the effective detection of 2-NA in real river water samples, revealing good recovery results.