In order to determine the content of nitrogen in culture medium at different culture time and compare the nitrogen use efficiency of various plantlets in vitro, near infrared spectroscopy technique and image analysis technique with multi-references were used to obtain the successive information of nitrogen consumption. The models between spectral information and nitrogen content in the medium were established via the partial least squares method together with artificial neural networks. They were used to predict the content of nitrogen in nine test samples. The result showed that the nitrogen use efficiency of incised leaf-type Atractylodes lancea was higher than that of long lanceolate leaf-type, and that of Orychophragmus violaceus was lower than that of Brassica napus. It suggested that the physiological function of the incised Atractylodes lancea was better than that of the long lanceolate.