The design of novel alternatives aimed at promoting sustainable agriculture is essential for counteracting the significant demand to feed an ever-growing population. The amphiphilic properties of zein make it an excellent biomaterial for the development of prolonged-release fertilizer. The objective of this work was to prepare and characterize phosphate (Ca-(HPO)·HO) release particles based on zein that can be used in agriculture to reduce the loss of this nutrient through runoff and leaching. Zein nanoparticles were prepared by nanoprecipitation method using ethanol (70% v/v). A 3 × 3 × 3 factorial design was used for phosphate-loaded zein nanoparticles. The zein-phosphate nanoparticles with zein 3% (w/v), poloxamer 188 0.15% (w/v), and phosphate 1 and 2% (w/v) were significantly the smallest in size (367 ± 16 nm and 326 ± 15 nm, respectively), the polydispersity index for both concentrations of Ca-(HPO)·HO was 0.3 ± 0.1, and there was a high z-potential on both nanoparticles (79.8 ± 11.6 mV and 178.8 ± 9.9 mV for nanoparticles with 1 and 2% w/v of Ca-(HPO)·HO). Fourier Transform Infrared Spectroscopy analysis shows that hydrogen bonds are the mechanism by which the PO groups are joined to the nanoparticles, and the deconvolution of the amide I shows the beta sheets are the predominant secondary structure for nanoparticles with 2% phosphate. Finally, the release of phosphate was close to 20%. In this work, it was possible to find favorable conditions for the formation of zein nanoparticles loaded with monobasic phosphate monohydrate by nanoprecipitation.