Objective: Our study aimed to identify key splicing regulatory sites within the 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 focus on intronic variations that may influence pre-mRNA splicing. The variations were filtered through a comparative analysis based on sequence conservation and associated disease severity. Variations located within highly conserved loci that are 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 capillary electrophoresis. For those in-frame aberrant splicing products, we assessed protein expression, protein secretion, and in vitro coagulation activity via Western Blotting (WB) and activated partial thromboplastin time (APTT). Results: (1) 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, it was further established that computational predictions have limitations and cannot accurately forecast specific splicing patterns or their proportions. (2) The classic GT-AG splice site on the intron was confirmed to be conserved. Additionally, it was observed that +5G at the donor end facilitates correct recognition of the splicing site, whereas the relatively conserved +7A does not play a significant regulatory role in this recognition process. Furthermore, deep intronic variants mimicking the classic splice donor "AG" motif cause misalignment of splice site recognition, significantly increasing the proportion of abnormal splicing. (3) 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—p.D93-G125delinsG and p.G94-D131del—and those of the wild-type FIX (P<0.01). Conclusion: (1) 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. (2) 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.