To investigate the physiological and behavioral comfort of drivers traversing urban underpass tunnels with various spatial structure conditions, a driving simulator experiment was conducted using 3DMAX and SCANeRTM studio software. Three parameters, including the slope, slope length, and height of a tunnel, were selected as research objects to explore the optimal combination of structural parameters in urban underpass tunnels. The heart rate (HR), interbeat (RR) interval, speed, and lane centerline offset value were collected for 30 drivers. Then, a measurement model of the relationship among HR, RR interval, speed, lane centerline offset value, and structural parameters was established by using partial correlation analyses and the stepwise regression method. On this basis, a structural constraint model based on the drivers’ physiological and behavioral comfort thresholds was also constructed. The results show that the driver’s HR, RR interval, speed, and lane centerline offsets are significantly related to the tunnel height, slope, and slope length. More importantly, this paper not only analyzed the effects of various structural parameters on drivers’ physiology and behavior but also proposed an optimized combination of structural parameters based on drivers’ physiological and behavioral comfort. It can reasonably improve tunnel design in China, ensure tunnel traffic safety, and seek the maximum comfort of the driver in the driving process.