The synaptic cleft is the space through which neurotransmitters convey neural information between two synaptic terminals. This space is presumably filled with extracellular matrix molecules involved in synaptic function or differentiation. However, little is known about the identities of the matrix components, and it remains unclear how these molecules organize the matrix in synaptic clefts. In this study, we identified Hasp, a Drosophila secretory protein containing CCP and WAP domains. Molecular genetic analysis revealed that Hasp diffuses extracellularly and is predominantly captured at synaptic clefts of cholinergic synapses. Furthermore, Hasp regulates levels of DLG and the nAChR subunits Dα6 and Dα7 at postsynaptic terminals. Hasp is required for trapping of another matrix protein, Hig, which is also secreted and diffused in the brain, at synaptic clefts of cholinergic synapses; however, Hig is dispensable for localization of Hasp at synaptic clefts. In addition, in the brains of triple mutants for the nAChR subunits Dα5, Dα6, and Dα7, the level of Hig, but not Hasp, was markedly reduced in synaptic regions, indicating that these nAChR subunits are required to anchor Hig to synaptic clefts. High-resolution microscopy revealed that Hasp and Hig exhibit segregated distribution within individual synaptic clefts, reflecting their differing roles in synaptogenesis. These data provide insight into how Hasp and Hig construct the synaptic cleft matrix and regulate the differentiation of cholinergic synapses, and also illuminate a previously unidentified architecture within synaptic clefts. The synapse has been extensively studied because it is essential for neurotransmission. By contrast, the space between the synaptic terminals, the synaptic cleft, is still an undeveloped research area despite its ubiquity in synapses. In fruit fly brains, we obtained evidence that the matrix protein Hasp and the previously identified Hig, both of which are secreted extracellularly, localize predominantly to synaptic clefts of cholinergic synapses, and modulate the levels of nAChR subunits on postsynaptic membranes. However, Hasp and Hig play differential roles in matrix formation and exhibit segregated distribution within synaptic clefts. These results reveal the molecular mechanisms of synaptic matrix construction and illuminate a molecular architecture within synaptic clefts previously unrevealed in any animal species.