Objective：To identify key splicing regulatory sites within introns of the F9 gene（the causative gene for hemophilia B） through the characterization of splice site consensus motifs and systematic screening for pathogenic variants in these regions. Methods： Based on the variations documented in the Factor Ⅸ Variant Database，we primarily focused on intronic variations that may influence pre-mRNA splicing. These variations were filtered through a comparative analysis based on sequence conservation and were associated with the severity of the disease. Variations located within highly conserved loci that were linked to severe hemophilia were selected as target variations and subjected to subsequent splicing predictions using three independent tools separately. The prediction results were then validated through minigene splicing assays，and specific splicing patterns were further investigated using denaturing capillary electrophoresis. For those in - frame aberrant splicing products，we assessed protein expression，protein secretion，and in vitro coagulation activity by Western blot and activated partial thromboplastin time. Results：We identified 15 variants located near the splicing site of exon 4 in the F9 gene as target variants through sequence and variant analysis. Minigene splicing assay confirmed that 14 of these variants could lead to abnormal splicing. In comparison with the bioinformatic prediction results，we further established that computational predictions exhibited limitations and could not accurately predict specific splicing patterns or their proportions. The classic GT-AG splice site on the intron was confirmed to be conserved. Additionally，we observed that +5G at the donor end facilitated correct recognition of the splicing site，whereas the relatively conserved +7A did not play a significant regulatory role in this recognition process. Furthermore，deep intronic variants mimicking the classic splice donor“AG”motif caused misalignment of splice site recognition，significantly increasing the proportion of abnormal splicing. Results from protein expression and activity analyses indicated notable differences in antigen synthesis and in vitro coagulation activity status between the two abnormal splicing variants of p.D93-G125delinsG and p.G94-D131del and those of the wild-type FⅨ（P ＜ 0.01）. Conclusion：The splice site（GT-AG）is pivotal in determining precise splicing. The less conserved +5G at the consensus region of the splice donor site in the intron 4 of the F9 gene also has a regulatory effect on splicing. Summarizing the differences in splice patterns caused by diverse variations can provide a solid theoretical basis for big data analysis and improve the accuracy of bioinformatics predictions.