The content and density of traffic signs directly affect the operation of urban road traffic and the acceptance of drivers. In order to make up the limitation of quantitative research on density threshold of traffic signs on urban roads, a real vehicle experiment was used to record the drivers’ psychophysiology characteristics. The psychological and physiological indexes of drivers, such as pupil area, fixation intensity, heart rate change rate and heart rate variability, were explored and then principal component analysis was used to present a new index S to represent driving visual comfort level, which was divided into 5 grade scales. The information entropy theory was applied to quantify the amount of information for road traffic signs in driving tests, and a regression relationship between the information content of traffic signs and the comfort index S was established. To meet the requirements of driving comfort, the visual psychophysiological load threshold was -2.289≤S<-1.526 for the level of very comfortable, 1.526≤S<-0.763 for the level of relatively comfortable, -0.763≤S≤0.763 for the level of comfortable, 0.763