Under sulfur (S) deficiency, crosstalk between nutrients induced accumulation of other nutrients, particularly molybdenum (Mo). This disturbed balanced between S and Mo could provide a way to detect S deficiency and therefore avoid losses in yield and seed quality in cultivated species. Under hydroponic conditions, S deprivation was applied to Brassica napus to determine the precise kinetics of S and Mo uptake and whether sulfate transporters were involved in Mo uptake. Leaf contents of S and Mo were also quantified in a field-grown S deficient oilseed rape crop with different S and N fertilization applications to evaluate the [Mo]:[S] ratio, as an indicator of S nutrition. To test genericity of this indicator, the [Mo]:[S] ratio was also assessed with other cultivated species under different controlled conditions. During S deprivation, Mo uptake was strongly increased in B. napus. This accumulation was not a result of the induction of the molybdate transporters, Mot1 and Asy, but could be a direct consequence of Sultr1.1 and Sultr1.2 inductions. However, analysis of single mutants of these transporters in Arabidopsis thaliana suggested that other sulfate deficiency responsive transporters may be involved. Under field conditions, Mo content was also increased in leaves by a reduction in S fertilization. The [Mo]:[S] ratio significantly discriminated between the plots with different rates of S fertilization. Threshold values were estimated for the hierarchical clustering of commercial crops according to S status. The use of the [Mo]:[S] ratio was also reliable to detect S deficiency for other cultivated species under controlled conditions. The analysis of the leaf [Mo]:[S] ratio seems to be a practical indicator to detect early S deficiency under field conditions and thus improve S fertilization management.