Background and aims <p>Stable hydrogen and oxygen isotopes are sensitive to soil moisture dynamics, making them crucial indicators for tracing ecohydrological cycles within the soil–plant-atmosphere continuum (SPAC). Biocrusts, prevalent in dryland ecosystems, critically regulate soil water balance within the SPAC. However, biocrusts’ ecohydrological functions remain controversial, especially regarding the processes causing water isotope fractionations. Thus, isotope analyses provide a promising approach to clarify the ecohydrological role of biocrusts.</p> Methods <p>We collected samples of biocrusts-covered and bare soils over two years, analyzing the dynamics of stable hydrogen (²H) and oxygen (¹⁸O) isotopes within soil water, rainwater, and dew.</p> Results <p>We found that δ²H and δ¹⁸O within surface soil (0–5&#xa0;cm) sensitively responded to rainfall and air temperature fluctuations. Compared to bare surface soil, biocrust cover enriched δ²H and δ¹⁸O by 7.4‰ and 1.5‰, respectively, indicating intensified soil water fractionations. Similar biocrusts-induced isotope enrichments were observed across the 0–50&#xa0;cm soil profile, which was simultaneously correlated with elevated soil moisture and temperature. We also found that the majority (86.5%) of unfractionated water in the uppermost biocrusts-covered soil (0–5&#xa0;cm) was derived from the subsurface 5–10&#xa0;cm depths, while uppermost bare soil water was mainly derived from 5–20&#xa0;cm depths.</p> Conclusions <p>Our findings highlight the critical roles of biocrusts in intensifying soil evaporation and non-rainfall water deposition, preventing deep rainwater infiltration, and increasing the contribution of 5–10&#xa0;cm subsurface water vapor to uppermost soil water, which advance our understanding of biocrusts’ role in ecohydrological processes of drylands.</p>