Two-way two-lane roads have a significant impact on road transportation infrastructure. Platoon formation on two-lane roads is one of the factors that affect the quality of traffic flow on two-lane roads. More specifically, the creation of a platoon increases the density of vehicles and the number of overtaking maneuvers and decreases the traffic performance of two-lane roads. The present study made an effort to investigate the effect of the platoon characteristics on the traffic flow of vehicles on two-lane roads. Moreover, it strived to develop the nonlinear regression model with a new approach to capacity calculation. Finally, a method for estimating the level of service (LOS) based on the number of followers per capacity (NFPC) and LOS classification using the KNN method was presented. Considering these aims, first, the relevant variables (which were related to platoon) including time headway (Ht), average travel speed (ATS), platoon size (PS), average platoon speed (APS), percentage of heavy vehicles (HV), percent time spent following (PTSF), number of overtaking (NO), density (ρ), and traffic flow were investigated on the examined roads. The results showed that the speed and the Ht were the most effective and the least effective platoon characteristics, respectively. Moreover, it was accompanied by the increase in PTSF which resulted in the increase in overtaking maneuver. Finally, the results regarding the developed model showed that the NFPC measure was able to predict the traffic flow of two-lane roads in a more satisfactory way compared to the two criteria, namely, PTSF and ATS in the Highway Capacity Manual (2016) since it increased ATS by 65% and decreased the capacity of two-lane roads by 21%.