Biological soil crusts (biocrusts) are associations of highly specialized communities and topsoil particles and play important ecosystem functions in drylands. Biotic interactions affected the assemblage of biocrust communities and thus their ecological function and adaption abilities. As the dominant organism in the moss crust, moss plays a significant role in modifying the microenvironment and releasing carbon, and this may affect the distribution and metabolic activity of diazotrophic cyanobacteria, which are the dominant diazotrophs. In this study, we examined the contribution of diazotrophic cyanobacteria to nitrogenase activity (NA) by investigating the NA under different light conditions and estimated the proportion of diazotrophic cyanobacteria from total diazotrophs. We tested the spatial distribution of cyanobacteria using laser confocal fluorescence microscopy (CLSM). Additionally, we separated diazotrophs in moss crust into free-living and moss-associated diazotrophs and compared the NA, nifH gene abundances and their transcripts, and the community composition of free-living and moss-associated diazotrophs. The light-dependent characteristic of NA and dominance of heterocystous cyanobacteria (accounting for over 90% of total nifH sequences at both the DNA and transcript levels) suggested a significant contribution of heterocystous cyanobacteria to NA. Cyanobacteria were mainly distributed in a few millimeters of subsoil in which abundant cyanobacteria were associated with moss rhizoids. The NA rates of moss-associated diazotrophs were comparable to those of moss crust and approximately 4–8 times higher than those of free-living diazotrophs, indicating the major role of moss-associated diazotrophs in nitrogen fixation. The community composition of moss-associated diazotrophs was similar to that of free-living diazotrophs. Moss-associated diazotrophs have a higher NA per copy of the nifH gene and transcript than free-living diazotrophs, proving the synergistic association of moss and cyanobacteria with NA.