Providing bicyclists favorable and comfortable riding environments is an important objective for transportation professionals. This study investigated the factors that contribute to bicyclists' perception of comfort on physically separated bicycle paths and quantified their impacts. A survey was conducted on 29 physically separated bicycle paths in the metropolitan area of Nanjing, China. A factor analysis and an ordered probit model were used to analyze the data. The results showed that the mean perception of comfort was significantly different between age groups, but not significantly different between gender groups and between users of electric bicycles and conventional bicycles. The model performance indicated that the ordered probit model with factor analysis addressed the correlation between independent variables and outperformed the ordered probit model without factor analysis. The model estimates showed that bicyclists' perception of comfort on physically separated bicycle paths was significantly influenced by physical environmental factors, including the width of the bicycle pathway, the width of the shoulder, the presence of grade, the presence of a bus stop, the surrounding land use, and the flow rate of electric and conventional bicycles. The findings yielded insights into bicyclists' psychological perception of comfort on physically separated bicycle paths. This research can help inform the design and planning of these facilities.