In order to explore stable route safety evaluation indicators, this study discusses the relationship between the heart rate variability (HRV) and other factors, such as design speed, operating speed, acceleration rate, and velocity gradient through a lot of experiments. The results show that operating speed coordination and velocity gradient (Gv) outperform others as road safety indicators. Speed coordination evaluation criteria:|ΔV85|≤10 km/h, good coordination; 10 km/h≤|ΔV85|≤ 20 km/h, general coordination, route indicators of adjacent sections should be adjusted, so that the speed difference is not more than 10 km/h;|ΔV85|≥20 km/h, poor speed coordination, it needs to readjust the design of the adjacent sections. Speed gradient evaluation criteria:Gv≤0.10, good road safety;0.10≤Gv<0.15, common road safety;Gv>0.15, poor road safety and the adjacent sections need to readjust. The conclusions provide the theoretical reference for highway safety evaluation.