Production of biodiesel represents a way to attain economic growth by increasing and securing energy supply for the developing countries, and it can also create job opportunities and an attractive source for the farmers. The research aims to utilize the Citrullus Colocynthis (L.) plant as a valuable alternative for producing biodiesel. The attention was drawn to the Citrullus Colocynthis (L.) due to the plant having the advantage of needing less water compared to other plants. Also, the Citrullus colocynthis plant can be planted in different climate conditions, so it has a good impact on the production capacity. Furthermore, the use of p-p-toluene-4-sulfonic acid monohydrate (PTSA) as an acid catalyst for pretreatment of Citrullus Colocynthis (L.) is the first time to use the acid as a catalyst with Citrullus Colocynthis (L.) oil to produce biodiesel. Citrullus colocynthis (L.) plant seeds were collected from western and northern Sudan; the oil was extracted through screw press extraction using an oil extractor; and the Citrullus colocynthis oil (CCO) was treated to reduce the free fatty acid (FFA) contents before starting the transesterification reaction using 0.3% p-toluene-4-sulfonic acid monohydrate (PTSA) as an acid catalyst. FFA was reduced to 0.3%, and FFA conversion was 91.76%. The final biodiesel produced was found to be 98.51%. The physiochemical properties of biodiesel were flash point 228°C, measured by the Seta Multiflash Cleveland Flash Point Tester; kinematic viscosity at 40°C, 5.094 cSt, measured by the viscometer (Petro Test Instrument Model TV400); pour point, -30°C, measured by the Cloud and Pour Point Test Cabinet (Norma Lab Analysis Instrument Model P592-France); and density at 150°C, 0.878 gm/cm³, measured by the Digital Density Meter (Petro Instrument Model DMA4500). The results attained in this study conformed to the international standard specifications for biodiesel fuel. The results of this study show that the Citrullus Colocynthis (L.) oil has the potential for use as an industrial feedstock for biodiesel production. PTSA can be used as a catalyst in the pretreatment of CCO via esterification reaction, where it showed very high catalytic activity to reduce the free fatty acids (FFA) content in the CCO; also, it has less environmental impact due to its ease of recoverability and reusability.