Macroscopic traffic flow variables estimation is of fundamental interest in the planning, designing and controlling of highway facilities. This article presents a novel automatic traffic data acquirement method, called MOM-DL, based on the moving observer method (MOM), deep learning and YOLOv3 algorithm. The proposed method is able to automatically detect vehicles in a traffic stream and estimate the traffic variables flow q, space mean speed vs. and vehicle density k for highways in stationary and homogeneous traffic conditions. The first application of the MOM-DL technique concerns a segment of an Italian highway. In the experiments, a survey vehicle equipped with a camera has been used. Using deep learning and YOLOv3 the vehicles detection and the counting processes have been carried out for the analyzed highway segment. The traffic flow variables have been calculated by the Wardrop relationships. The first results demonstrate that the MOM and MOM-DL methods are in good agreement with each other despite some errors arising with MOM-DL during the vehicle detection step due to a variety of reasons. However, the values of macroscopic traffic variables estimated by means of the Drakes’ traffic flow model together with the proposed method (MOM-DL) are very close to those obtained by the traditional one (MOM), being the maximum percentage variation less than 3%.