On the basis of the result of differential proteome study during the anthers development in wolfberry variety ‘Ning Qi No.1’ and its male sterile mutant ‘Ning Qi No.5’, we have isolated an anther development related gene Lb14-3-3b, and confirmed the gene is dominant expressed in floral organs. In order to further understand its expression characteristics in floral organs and functions related development of floral organs, we use real-time quantitative PCR approach to analysis Lb14-3-3b gene expression level at different stages of wolfberry anther development. At the same time, we constructed plant over-expression vector Lb14-3-3b-pCambia1305.1-35s and transformed it into model plant tobacco by method of Agrobacterium tumefaciens mediated. The results showed that Lb14-3-3b was expressed during all stages of the wolfberry anther development and the expression level highest at the stage of two-nuclear pollen grain. Compared with wild type, transgenic tobacco plants grew relatively slow and dwarf, floral organ distortion, such as one petals and stamen absent, four angle flower patterns. It is deduced that Lb14-3-3b gene might play a regulatory role in the process of tobacco plant growth and its floral organ development. It still needs to study on the growth and development of wolfberry to understand the regulated mechanism of 14-3-3 proteins. The results provide a reference for the further explore the regulation of the gene in the growth and development of Lycium barbarum L.