A large number of long-and large-scale highway tunnels were built due to the high mountains and large rivers in Chongqing. While safety accidents frequently occurred during the operation period. In order to explore the regularity and characteristics of accidents, and to clarify the key factors affecting the operational safety, 260 accidents were investigated and analyzed in this paper. The results show that the number of accidents is positively correlated with the length of the tunnel, and in the case of similar accidents, the casualties of long tunnels are more serious than those of extra-long tunnels, accounting for 71% of the fatalities. Traffic accidents account for 87% of the total accidents during the operation period, and the main form is “vehicle-vehicle” accidents such as rear-end accidents and collisions, which account for 51%. Minibuses and trucks are the main types of traffic accidents, accounting for 57.3% and 38.2%, respectively. Motorbuses have the highest casualty rate, with 4 persons per case. Traffic accidents mainly occur from January to February, April to August and October, while 80% of non-traffic accidents occur in spring and summer. The most unfavorable combination of casualties is the traffic accident caused by truck in summer (July) from 10:00 to 16:00. According to the regularity and characteristics of accidents, basic reference for operation safety management of long-and large-scale highway tunnel in Chongqing can be provided from this paper.