Aging railroad bridges present complex challenges due to advancing deterioration and outdated design assumptions. This study develops a comprehensive analytical approach for assessing an aging steel truss railroad bridge through finite element (FE) modeling, sensitivity analysis, and model updating, supported by field testing. An initial FE model of the bridge was created based on original drawings and field observations. Field testing using a laser Doppler vibrometer captured the bridge’s dynamic response (vibrations and deflections) under regular train traffic. Key structural parameters (material properties, section properties, support conditions) were identified and varied in a sensitivity analysis to determine their influence on model outputs. A hybrid sensitivity analysis combining log-normal sampling and a genetic algorithm (GA) was employed to explore the parameter space and calibrate the model. The GA optimization tuned the FE model parameters to minimize discrepancies between simulated results and field measurements, focusing on vertical deflections and natural frequencies. The updated FE model showed significantly improved agreement with observed behavior; for example, vertical deflections under a representative train were matched within a few percent, and natural frequencies were accurately reproduced. This validated model provides a more reliable tool for predicting structural performance and fatigue life under various loading scenarios. The results demonstrate that integrating field data, sensitivity analysis, and model updating can greatly enhance the accuracy of structural assessments for aging railroad bridges, supporting more informed maintenance and management decisions.