Background: Small non-coding RNAs are essential regulators of gene expression at the transcriptional and posttranscriptional levels. High-throughput sequencing has revealed thousands of predicted small RNAs; however, only a few of these have been well characterized. Northern blotting is the most convincing method for small RNA validation. Findings: In this study, we improved the Northern blot method by using biotin-labeled probes. miRNAs and siRNAs derived from both Arabidopsis thaliana and Oryza sativa were investigated. The results suggest that this improved method is sensitive and efficient, with approximately 5 mu g of total RNA being sufficient for detection. Furthermore, long-term storage of probes labeled in this manner is more convenient, less contaminative and degradative compared with traditional probes. Conclusions: This protocol is an alternative strategy for small RNA detection and represents an efficient means of researching small RNAs.