This paper investigates the incipient motion of sediment particles under non-uniform flow in river and laboratory. In rivers, the non-uniform flow is often observed due to the presence of various bed forms. Threshold condition has been examined by using the Shields diagram based on the uniform flow assumption, however, this approach can be led to fallacious results for non-uniform flows where the effect of pressure gradient is significant due to bed forms. This study investigates the chronological order of incipient motion of the particles, the average threshold velocity (Ucr), and Shields parameter for non-uniform flows. River data collection with gravel is used for investigating the incipient motion of surface layer of river bed and the laboratory data collection is considered studying the incipient motion of sub-surface layer of river. Both river and laboratory data collections are conducted in the presence of bed forms. Results reveal that the Shields diagram underestimates the particle incipient motion under accelerating and decelerating flows for the both case of laboratory and river. In both weak and general motion in the laboratory, the values of the critical Shields parameter are located below the Shields diagram, showing no particle motion. Our analysis shows that the incipient motion in river is affected by the presence of bed forms, river width changes, and flow non-uniformity conditions. The results show that in the accelerating flow (the bed form exit with a negative slope), the incipient motion is greater than the decelerating flow (the bed form entrance with a positive slope).