The best condition for the biodiesel synthesis process from a non-edible and renewable source, custard apple seed oil, is presented in this paper, as well as its characterization. The seeds were harvested from Ethiopia's Jimma zone, and their average moisture, ash, fiber, protein, and oil contents are 5.7, 3.9, 2.1, 19.8, and 37.2 percent, respectively. Soxhlet and hexane as a solvent were used to extract the oil. Trans-esterification of oil with methanol and sodium hydroxide (NaOH) as a catalyst for biodiesel generation was investigated. The molar ratio of methanol to oil, temperature, catalyst, and reaction time all had a significant (p<0.05) impact on biodiesel yield. The optimum process parameters coupled to obtain the highest biodiesel output were reaction temperature of 72.426°C, catalyst 2.002wt %, methanol: oil molar ratio 12.334, and reaction time of 84.010min, according to the Experimental Design Response Surface Methodology (RSM) and ANOVA analysis. With a desirability of 1, the model anticipated a yield of 89.230±0.28 percent. Acid value, Saponification value, Kinematic viscosity, Density, Heating Value, Fire Point, and Flash Point are all physicochemical parameters of biodiesel made from custard apple seed oil (FP) 1.12 mg KOH/g, 16.75 mg KOH/g, 5.67mm2/s, 894.5kg/m3, 42.072kJ/kg 134oC and 179oC satisfied the ASTM criteria for biodiesel, respectively. In general, this research supports the manufacturing of biodiesel from Custard Apple seeds (Annona squamosa) oil as a means of replacing non-renewable energy with renewable energy and increasing the value of custard apple fruit.