MicroRNAs (miRNAs) are single-stranded noncoding small RNAs, frequently expressed as clusters. The co-expressed miRNA clusters are pivotal in coordinately regulating multiple processes, including embryonic development, cell cycles and cell differentiation. In the study, relative expression analysis of miRNA cluster was performed based on isomiRs from high throughput DNA sequencing data using SOLiD2122; system. Similar expression pattern of miRNA cluster was detected by analyzing the most abundant isomiR and multiple isomiRs, respectively. According to all the multiple isomiRs, miRNA* species always had fewer sequence counts and indicated different expression patterns with their miRNAs. Although co-transcribed from a single promoter as a single polycistronic transcript, miRNAs in gene cluster showed distinct levels. Inconsistent expression levels also could be detected after normalization of sequence counts despite degree of divergence was largely reduced. The difference of expression pattern might result from miRNA half-life because of degradation degree and rate. Inconsistent expression levels of members in miRNA cluster maybe contribute to complex biological processes in particular developmental contexts at specific times. The expression patterns according to the most abundant isomiR and all the multiple isomiRs are consistent, and relative expression analysis based on sequence counts and isomiRs from high throughput sequencing data provides a feasible method to analyze different miRNAs.