Abstract: Biochar amendments can modify fertilizer nitrogen (N) availability in soil and crop N uptake. However, hobiochar addition affects crop N uptake and fertilizer N recovery under various N levels is not yet well understood. To address this question, we conducted a two-season [cotton (Gossypium hirsutum L.) -barley (Hordeum vulgare L.) rotation] pot experiment that included four N fertilizer rates (0, 75, 150, and 300 kg N ha-1, supplied as urea-15N) combined with two straw-biochar rates (0 and 15 t ha−1). Soil properties, plant root morphology, N uptake, and biomass yield were studied. Biochar addition decreased soil inorganic N content but increased urea-N retention at cotton harvest, leading to 32% of the applied urea-N accumulating in soil compared with 27% without biochar, averaged across fertilizer N rates. Use of 15N fertilizer showed that biochar increased plant uptake of indigenous soil N, not fertilizer N. An obvious decrease in urea-15N recovery induced by biochar was observed at 75 kg N ha-1, but not at 150 or 300 kg N ha-1. The efficiency of urea-15N recovery by plants (15NRE, 34-45%), measured using the tracer method, was much lower than that measured using the traditional non-isotope method (NRE, 67-96%).