Climate change is a major issue all around the world. The transportation industry currently accounts for most CO₂ emissions. The goal of this research is to develop a real-world eco-driving cycle for internal combustion engine motorcycles that can reduce fuel consumption and CO₂ emissions. This study developed onboard measuring equipment to measure the speed profile and fuel consumption of a motorcycle driving in real time. A total of 78 motorcycle riders rode a test motorcycle with the onboard equipment along a road network to collect real-world data. All of the collected real-world data were analyzed by cluster analysis based on fuel consumption (km/L) to divide riders into two groups, high-fuel-consumption riders and low-fuel-consumption riders. The collected real-world data of the low-fuel-consumption riders were used to develop a real-world eco-driving cycle, whereas the collected real-world data from the high-fuel-consumption riders were used to develop a real-world non-eco-driving cycle. The CO₂ emissions were calculated by the speed profiles of the developed driving cycles. The findings reveal that the real-world eco-driving cycle provided a fuel consumption rate 39.3% lower than the real-world non-eco-driving cycle. In addition, the real-world eco-driving cycle provided a CO₂ emission rate 17.4% lower than the real-world non-eco-driving cycle. The application of the developed real-world eco-driving cycle for motorcycles is proposed.