Objective To isolate and identify canine parainfluenza virus (CPIV) from the a dog suspected of CPIV infection, then to analyze the gene sequence variation of N,HN and F genes and provide a molecular biology basis for the diagnosis, treatment, prevention and control of CPIV infection. Methods Samples of the lung tissue was taken from a dead dog suspected of CPIV infection, and were inoculated into Vero cells. CPE were observed after three blind passages, then the cell culture fluid after 72-hour-cultuere was assayed for further PCR identification, blood coagulation characteristics and morphological observation. Moreover, 3 pairs of oligonucleotide primers were designed, and N, HN and F complete genes of CPIV were amplified by RT-PCR from positive CPIV culture fluid. The genetic variation of the three genes were further analyzed by biological software, and phylogenetic trees were produced. Results One strain of CPIV was isolated and named QF20100726. The results showed that the CPIV strain could agglutinate Guinea pig, pig,chicken and human o type blood cells,and had the basic ultrastructural chatacteristics of parainfluenza virus. Compared with 10 representative CPIV genes, N, HN and F gene phylogenetic analysis of the QF20100726 CPIV gene showed 95.7% to 99.8%, 94.7% to 99.6% and 94.7% to 99.6% nucleotide identity, respectively, and 97.4% to 99.6%, 96.3 to 99.6%, 95.6% to 99.3% amino acid identity, respectively. Of these aa substitutions, 2 substitutions (V208A, A301T) occurred in the N open reading frame (ORF), and 2 substitutions (T56S, T89M) occurred in the F open reading frame (ORF). Conclusions One strain QF20100726 of CPIv is successfully isolated, and the phylogenetic trees of N, HN and F genes from the QF20100726 CPIV strain show close phylogenetic relationship with other CPIV isolates. The data provide a valuable molecular biology basis for the studies on prevention and control of CPIV infection.