Plantlets grown in vitro with a mixed nitrogen source utilize sucrose and CO2 as carbon sources for growth. However, it is very difficult to obtain the correct proportion of assimilated nitrate, ammonium, sucrose and CO2 for plantlets. Consequently, the NH4+/NO3- use efficiency for carbon fixation derived from the assimilation of sucrose/CO2 is still unclear for plantlets. To reveal the coupled relationship of carbon and nitrogen assimilation in plantlets, the bidirectional stable nitrogen isotope tracer technique was employed to quantify the proportions of nitrate and ammonium utilized at different NH4+/ NO3- ratios, and the proportions of sucrose and CO2 assimilation were quantified by the foliar δ13C values of plantlets. There was an obvious difference in the assimilation of nitrate and ammonium under different NH4+/NO3- ratios for Brassica napus (Bn) plantlets. The proportion of sucrose/CO2 assimilation depended on the NH4+/NO3- ratios of the Bn plantlets. Both nitrate and ammonium assimilation were independent of sucrose/CO2 assimilation. Based on the proportion of CO2, sucrose, nitrate and ammonium utilization, the efficiency of nitrate/ammonium use (as indicated by the C/N ratio) for carbon fixation derived from the assimilation of sucrose/CO2 can be quantified for Bn plantlets.