The plume-chasing method has shown great advantages in measuring on-road emission factors (EFs) compared with regulatory methods like dynamometer and portable emission measurement systems (PEMS). In this study, a new on-board measurement system incorporating ultrasonic anemometers and solid-state Lidar was developed to investigate the uncertainties of on-road emission factors measured by plume-chasing method due to variables such as on-road wind velocity, chasing speed, chasing distance, and turbulent kinetic energy (TKE). A series of PEMS-chasing experiments for heavy-duty diesel vehicles (HDDVs) were conducted on both highways and local roadways in Beijing, China. Our analysis demonstrated that the differences in EF estimations between concurrent plume-chasing and PEMS measurement decreased with increasing chasing speed as a result of greater vehicle-induced TKE in the wake between HDDV and the mobile platform, whereas the effect of chasing distance on EF estimations appeared insignificant within the tested distance range (12–22 m). In the case of strong crosswinds, overprediction of chasing-based EFs was observed due to convective plume mixing from surrounding vehicular sources. The findings of this study contribute greatly to interpret emission factors measured by the plume-chasing method, and also calls for a future study to develop real-time EF correction algorithms for large-scale mobile chasing measurements.