The invention provides a eucaryon alternative splicing analysis method and system based on RNA-seq data. The method comprises the steps that transcriptome original sequencing data of one or more samples of a certain eucaryon with a reference genome and annotation is acquired through an illumina next-generation sequencing platform; unqualified data is filtered out, and the remaining data serves asto-be-analyzed data; next, basic analysis is performed, wherein the to-be-analyzed data of all the transcriptome samples is compared to the reference genome of the species, and a unique comparison result is screened out; expression quantities of all sample genes are calculated; the genes with significant difference expression are screened out; functional annotation and analysis are performed on the differential genes; then alternative splicing analysis is performed, wherein a known alternative splicing event is identified; a new alternative splicing event is identified; the difference betweenalternative splicing events of samples (sample groups) is analyzed; the correlation between alternative splicing and gene expression is analyzed; the alternative splicing analysis result is subjectedto statistical analysis, and a report is generated; and an alternative splicing visual graph is generated.