In recent years, the promotion of green travel and the emergence of bicycle sharing have dramatically increased the population of people traveling by bicycle in China. Bicycle sharing has become an indispensable daily means of travel for some people. Therefore, the ride comfort and safety of bicycle lanes should be paid more attention. Toward meeting the demand for smooth bicycle lanes, we propose a new method of testing the roughness of bicycle lanes based on the riding condition and explore the possibility of its realization. A riding profiler is assembled and designed, and its test repeatability is verified by repeated measurements. The riding profiler is also applied to different pavement types with various speeds for roughness testing. The results demonstrate that the speed and pavement type affect the vibration acceleration and vertical displacement during riding, and the greater the riding speed, the stronger the vibration sensation. In addition, the vibration is strongest when riding on concrete block pavement and weakest on dense asphalt pavement. On the basis of the test results, the acceleration interference is taken as an evaluation index to analyze riding comfort under different conditions.