Attainment of cycling comfort on urban roads encourages travelers to use bicycles more often, which has social and environment benefits such as to reduce congestion, air pollution and carbon emissions. Cycling vibration is responsible for the cyclists' perception of (dis)comfort. How asphalt pavement's surface characteristics relate to cycling comfort, however, remains undiscovered. In this study, the cycling vibration intensity on 46 sections of 24 urban roads was tested using a dynamic cycling comfort measure system while the cyclists' perception of vibration was identified via questionnaires; the cycling comfort was then defined based on the cycling vibration. To record the accurate pavement-tyre interface under a stable environment, a total of 19 pavement sections were scanned using a 3D digital camera. These 3D models were then 3D printed, which are used to conduct the pressure film test using a self-developed pavement-tyre interface test system. Three ranges of pressure films were adopted to characterize the pavement-tyre interface via 9 parameters, namely contact area (A c), unit bearing area (B u), stress intensity (S i), stress uniformity (S u), kurtosis (S ku), spacing (Sp a), maximum peak spacing (Sp max), radius ratio (R r) and fractal dimension (F d), in consideration of the area characteristics, pressure amplitude, peak spacing and shape of the interface. Finally, the significant interface parameters were identified, and the regression model between interface parameters and cycling comfort was established. Results show that the cycling vibration was described to be 'very comfortable' when the human exposure to vibration level (a wv) was less than 1.78 m/s 2 ; 'comfortable' when the a wv was between 1.78 m/s 2