Objective Driving comfort is crucial for tunnel safety because tunnel sections on freeways often introduce significant environmental changes that can compromise comfort and increase the risk of traffic accidents. This study aimed to quantitatively evaluate the driving comfort in tunnel sections and its implications for safety management. Methods Four indicators were used to assess the driving comfort: heart rate growth rate (Hrgr), skin conductance response (SCR), speed, and acceleration. The CRITIC weighting method was employed to calculate a quantitative driving comfort score, and the presence and severity of discomfort were used to evaluate the safety of each tunnel area. In addition, the evaluation was based on a naturalistic test consisting of Hrgr, SCR, speed, and acceleration data. A total of 32 participants were recruited based on a web-based questionnaire screening process, after which they were tested while driving through 30 tunnel sections on the roadway. These 30 tunnels included 14 short (< 500 m), 12 medium (500–1,000 m), and 4 long (1,000–3,000 m) tunnels. Results The results revealed that the four selected indicators exhibited minimal multicollinearity and effectively captured the driving comfort. Among the indicators, SCR had the most significant contribution to the driving comfort score. Most drivers did not experience substantial discomfort while driving through tunnels. The area where drivers were most susceptible to discomfort was the middle zones of tunnels. However, drivers were more likely to experience strong discomfort in the outside exit, entrance, and middle zones of short, medium, and long tunnels, respectively. Conclusions This study provides a comprehensive set of safety evaluation methods for tunnel sections on freeways, with a focus on quantifying the driving comfort. The findings provide theoretical support for freeway management personnel in implementing personalized controls in different tunnel areas with the aim of enhancing tunnel safety and mitigating the occurrence of traffic accidents.