The strain data acquired from structural health monitoring (SHM) systems of large-span bridges are often contaminated by a mixture of temperature-induced and vehicle-induced strain components, thereby complicating the assessment of bridge health. Existing approaches for isolating temperature-induced strains predominantly rely on statistical temperature–strain models, which can be significantly influenced by arbitrarily chosen parameters, thereby undermining the accuracy of the results. Additionally, signal processing techniques, including empirical mode decomposition (EMD) and others, frequently yield unstable outcomes when confronted with nonlinear strain signals. In response to these challenges, this study proposes a novel temperature-induced strain separation technique based on improved blind source separation (BSS), termed the Temperature-Separate Second-Order Blind Identification (TS-SOBI) method. Numerical verification using a finite element (FE) bridge model that considers both temperature loads and vehicle loads confirms the effectiveness of TS-SOBI in accurately separating temperature-induced strain components. Furthermore, real strain data from the SHM system of a long-span bridge are utilized to validate the application of TS-SOBI in practical engineering scenarios. By evaluating the remaining strain components after applying the TS-SOBI method, a clearer understanding of changes in the bridge’s loading conditions is achieved. The investigation of TS-SOBI introduces a novel perspective for mitigating temperature effects in SHM applications for bridges.