Over the past few years, incubated biocrusts (IBSC)—the inoculation of soil/sand with cyanobacteria, moss, and lichen—have become one of the most promising biotechnological strategies for preventing soil erosion and restoring soil function in degraded drylands. Soil nutrient content (C, N, and P) is one of the key factors that influences IBSC colonization and development; however, the effects of soil C:N, C:P, and N:P stoichiometric ratios on the colonization and development of IBSC in desert environments are little known. We used four soil substrates, collected from four areas on the southeastern edge of the Tengger Desert, China, to incubate biocrusts. The four substrates differed in particle size and nutrient content. We flattened the dunes so their sand surfaces were level before covering them with soil substrates. We used a fully factorial design with four soil substrates and with and without biocrust additions, generating a total of 12 different treatments. The soil substrates differed in C, N, and P content and C:N, C:P, and N:P stoichiometric ratios. We measured IBSC coverage and thickness to establish relationships between IBSC characteristics and soil C:N, C:P, and N:P stoichiometric ratios. After 12 months of development, all treatments had significantly more cyanobacteria coverage, lichen coverage, and total coverage of IBSC than did control plots, which had little or no IBSC development. C:N and C:P ratios were significantly positively related to cyanobacteria coverage and total coverage of IBSC. Soil C:N and C:P ratios were mainly controlled by soil C; C limitation was greater than N and P limitation. Our study indicates that increasing initial soil substrate C content to improve C:N and C:P ratios will help the recovery of biocrusts and IBSC colonization. We demonstrate that stoichiometric ratios of soil should be a concern when assessing IBSC restoration treatments and when using IBSC to restore degraded land, especially at large scales.