The objective of this study is to investigate the potential of functional near-infrared spectroscopy (fNIRS) combined with heart rate variability indices, for the evaluation of the mental workload of urban rail transit drivers under simulated driving conditions, particularly during task engagement and disengagement. Experienced metro drivers wearing fNIRS monitoring systems were asked to drive for 90 min in a professional metro driving simulator. Workload stimulus tasks were added and an n-back task (n = 3) was implemented to induce workload in the simulated driving experiment. Experimental results indicate that fNIRS are sensitive to mental workload and reliable for discriminating the degree of mental workload. Research findings demonstrate the feasibility and reliability of fNIRS as a tool for real-time evaluating and monitoring driver mental workload along with task factors from a perspective of brain activations during simulated or actual driving. Practitioner Summary: This study provides evidence for the potential of functional near-infrared spectroscopy (fNIRS) for the evaluation of the mental workload of urban rail transit drivers under simulated driving conditions. The first fNIRS application to mental workload evaluation in the field of urban rail transportation helps companies develop reasonable shiftwork schedule and ensure operation safety. Abbreviations: fNIRS: functional near-infrared spectroscopy; oxy-Hb: Oxy-hemoglobin; NASA-TLX: National Aeronautics and Space Administration Task Load Index; EEG: electroencephalogram; ECG: electrocardiogram; HRV: variability; LF: low-frequency power; HF: high-frequency power; PFC: prefrontal cortex; NIRS: near-infrared spectroscopy; DWT: discrete wavelet transform; EMG: electromyography; DT: determination test; TP: total power; LF(norm): standardized LF; HF(norm): standardized HF; VLF: very low frequency; deoxy-Hb: deoxy-hemoglobin.