The purpose of this study is to investigate the molecular characteristics of non-coding RNA617 in Salmonella enterica serovar Typhi (S. Typhi), as well as its effect on biofilm formation and its mechanism. Northern blot was used to detect the expression of ncRNA617; 5’RACE (5’-rapid amplification of cDNA end) and 3’RT-PCR (reverse transcriotion-polymerase chain reaction) were used to identify possible transcription initiation and termination sites. ncRNA617 knockout strain, complemented strain and overexpression strain of S. Typhi were constructed. The bacterial biofilm formation experiment was used to observe the effect of ncRNA617 on the formation of biofilm; qPCR (quantitative real-time PCR) was used to analyze the effect of ncRNA617 on the expression level of genes related to biofilm formation. Northern blot showed the presence of ncRNA617 with a length of about 300 nt; 5’RACE and 3’RT-PCR revealed that the 5’end of the transcript was located at 967 nt downstream of the mig-14 stop codon, and the 3’end of the transcript was located at 2378-2560 nt upstream of start codon of t2681. Biofilm formation experiment showed that compared with wild control strain, the biofilm formation ability of the ncRNA617 knockout strain was enhanced (P<0.05). The biofilm formation ability of the complemented strain was restored to the level of the wild strain, and the biofilm formation ability of the overexpression strain decreased (P<0.05). qPCR results showed that ncRNA617 negatively regulated the transcriptional expression levels of several biofilm formation related genes. Predicted by bioinformatics methods, it was found that ncRNA617 had different binding regions with regulated genes. It is concluded that ncRNA617 exists in S. Typhi and its length is about 270～452 nt. ncRNA617 may negatively regulate gene expression by targeting genes related to biofilm formation, and then negatively regulates the biofilm formation of S. Typhi.