Abstract: Boron (B) and magnesium (Mg) play a vital role in plant growth and secondary metabolites synthesis. To understand the effect of B and Mg deficiency on the biomass accumulation and leaf cannabinoid (CBD) concentration of hemp (Cannabis sativa L.), sand cultured seedlings of Yunma #7 and Jinma #1 were irrigated with nutrient solution deficient in B or Mg for four weeks. Plant morphology, biomass accumulation and CBD concentration in the leaves were determined. Under B deficient condition, the plants developed curly and yellowish new leaves, followed by shoot-tip necrosis and height stagnation. Four weeks after B deficient treatment, the plant height, stem diameter, and leaf dry weight were lower than those of control treatment (CK) by 35.2%, 12.5% and 11.1%, respectively, the CBD concentration in leaves was lower than that of CK by 55.0%. Under Mg deficient treatment, the plants showed firstly interveinal chlorosis on the lower leaves, then the symptom gradually appeared on the upper leaves. Four weeks after Mg deficient treatment, the plant height, stem diameter, and leaf dry weight were lower than those of CK by 9.3%, 12.5% and 13.9%, respectively, the concentration of CBD in the leaves did not differ significantly from that of CK. These results could support the diagnosis of B and Mg deficiency in hemp plants, and the increase of CBD yield through optimizing B and Mg management.