As a serine/threonine protein kinase family, CBL-interacting protein kinase (CIPK) is one of the most populated families. CIPKs are activated by calcineurin B-like protein CBLs and form the CBL-CIPK network which can play an important role with response to different adversity stress and impact on the process of plant growth and development. Although many research have done in Arabidopsis, but seldom studies have done in model plant tobacco. We have identified NsylCIPK3, one CIPK gene from Nicotiana sylvestris genome which is highly homolog with PtCIPK3 and AtCIPK3 up to 87.5% and 68.4% accordingly. Amino acid sequence analysis showed that, the NsylCIPK3 protein encoded a typical activation loop domains at N-terminal, and C-terminal has a highly conserved NAF domain. Phylogeny revealed that it belonged to number II subfamily of CIPK family with rich in introns. Expression pattern analysis of different tissue demonstrated that NsylCIPK3 can express in both root and leaf, especially highly expressed in the leaves of mature stage. Real-time quantitative PCR results displayed that it can response to high salt, high temperature, UV, low potassium and chilling treatments. The results of protein-protein interaction by yeast two hybrid illustrated that NsylCIPK3 interacts with NsylCBL9. It assumes that NsylCIPK3 can be activated by NsylCBL9 and perform as NsylCBL9-NsylCIPK3complex, this complex might be involved in abiotic stress signal transductions pathways in tobacco.