BACKGROUND: The in vitro cultivation of individual stages of the Plasmodium falciparum mosquito life cycle is notably challenging. The main difficulty is replicating the intricate nutrient and metabolite exchanges necessary for oocyst development and sporozoite (SPZ) formation in the three-dimensional environment of the mosquito midgut. Replicating these conditions is essential for understanding the biological interactions between mosquito and parasite, as well as advancing malaria vaccine development. METHODS: An in vitro three-dimensional system was developed that closely mimics the mosquito midgut epithelium, basal lamina, and haemolymph, facilitating the production of substantial quantities of haemolymph-like Pf SPZ. RESULTS: Use of an extracellular matrix-coated Alvetex(®) Strata scaffold, combined with optimized culture medium, supports efficient oocyst attachment and provides the necessary nutrients for robust production of haemolymph-like SPZ. This system enables full maturation of oocysts, as shown by immunofluorescence assays (IFA), and allows timely release of in vitro SPZ (IVS) between days 11 and 15, comparable to the in vivo mosquito timeline. Haemolymph-like SPZ generated were found to be infectious, as evidenced by successful HC04 infection in in vitro and in vivo studies using FRG-huHep mice. Similar outcomes were observed across different P. falciparum strains. CONCLUSIONS: Implementation of the Alvetex Strata scaffold, optimized medium, and improved ookinete production consistently enables in vitro generation of large quantities of haemolymph-like SPZ.