Bitter orange (Citrus aurantium) is a fruit that is grown in different regions of Iran. Moreover, bitter orange has also a lot of seeds that mostly considered as a waste part of the fruit. Bitter orange's seeds have a lot of oil content (similar to 38%), so the bitter orange seeds can be a suitable source for generating energy from wastes. In the current study, bitter orange seed oil (BOSO) uses as a novel feedstock for biodiesel production. BOSO has high acid value (16.58 mgKOH/g), so first of all, esterification reaction performed to reduce the acid value of the oil. Finally, the amount of acid value reduced to 1.2 mgKOH/g. In the next step, the transesterification reaction performed to produce biodiesel from bitter seed oil, and then the physicochemical properties of the produced biodiesel investigated. The results showed that the maximum biodiesel yield of 97% obtains in the transesterification reaction's condition of the reaction temperature of 60 degrees C, catalyst concentration of 1 wt%, rotational speed of 600 rpm, and molar ratio (methanol: oil) of 8:1. The results of investigating the physiochemical properties of the produced biodiesel show that all measured physiochemical properties of the bitter orange biodiesel can pass the ASTM standard requirements. According to the obtained results, we can conclude that bitter orange has an acceptable potential to introduce as a new resource for biodiesel production.