There is limited information on the influence of environmental and behavioural factors on cyclists’ risk perception. This study gives insights into the factors shaping cyclists’ perception of risk. Twenty-six cyclists were assessed for their psychological distress and previous cycling experience, following which their heart rate was monitored while cycling along a variety of urban routes. The findings are that cycling through commercial areas, one-way routes, and routes with speed limits reduced their perceived risk. However, wider cycling routes and bikeways were associated with an increased heart rate. This study implied that both individual and environmental characteristics significantly influence risk perception. Both the psychological well-being of cyclists and cycling infrastructure should be considered to promote urban cycling. Risk perception serves as an indicator to guide the development of urban cycling facilities, enabling urban planners and policymakers to promote healthier, more sustainable cities through effective promotion of cycling for urban commuting.