Deep sequencing of small RNAs (sRNA) is widely used in sRNAs studies in plants. In order to investigate the sequencing frequency variation of sRNAs, the same sRNA samples from rice grains were sequenced twice using deep sequencing technique. The sRNAs were classified into three categories, high abundance (> 100 RPM), medium abundance (10–100 RPM) and low abundance (1–10 RPM). According to the repeat sequencing data of the same sample, highly expressed sRNAs (> 100 RPM) were less subject to random drift, and 95% of the sRNAs Log2 ratio between two samples fell between -0.649 and 0.558. The same trend was observed in mediumly expressed sRNAs (10–100 RPM), and 95% of the Log2 ratio fell between -0.535 and 0.759. As to lowly expressed sRNAs (1–10 RPM), 95% of the Log2 ratio varied between -1.009 and 1.011. These results can be used as a theoretical guide to find differentially expressed sRNAs in sRNA studies in plants.