A crucial factor, route familiarity, can affect traffic safety. Nevertheless, focus on the influence of route familiarity on drivers’ eye fixations at urban intersections has received less attention. Identifying the real-world effect of route familiarity on drivers’ eye fixations at urban intersections in Changsha, China, was the objective of this study. Their visual fixation indicators were recorded while unfamiliar drivers and familiar drivers drove a 9 km-long route with nine intersections in an urban environment, but their effectiveness was indicated by the data collected 150 m before the lane stop and 50 m after the lane stop at these intersections. From the analysis of the extracted data, the results indicated that route familiarity could influence drivers’ processing times in the left window (LW) and other areas (OT). Compared with familiar drivers, unfamiliar drivers had longer processing times and higher mental workloads for the right front (RF). For the vehicle’s front (RF, FL, FR), the sampling rates and mental workloads of unfamiliar drivers were higher than those of familiar drivers, but it was the opposite for the driver’s sides (LW, RW) and rear (LM, RM, ReM). It was also indicated that the phenomenon said to increase familiarity with the route and make drivers more likely to be distracted in urban intersections had not been found. From the present findings, the effect of route familiarity on drivers’ eye fixations at urban intersections was confirmed. The high accident risk of familiar drivers could be partly explained by the decrement in drivers’ eye fixation strategies. However, the strategies could not account for the phenomenon that more familiar drivers are involved in rear-end accidents. Therefore, the reason can be investigated based on drivers’ visual scanning strategies, their physiological signals and driving behavior in the future.