MicroRNAs(miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and their aberrant expression occurs during the development of malignant diseases. Recently, miRNAs have been proposed as potential prognostic and predictive biomarkers for early diagnosis. However, a major obstacle in rapid miRNA analysis from real samples is the lack of ultrasensitive and quantitative techniques. In this regard, the use of chemiluminescence(CL) system offers a highly sensitive strategy for detecting miRNAs. In this article, an ultrasensitive approach has been established for the quantification of miRNAs, using magnetic beads(MBs) and alkaline phosphatase(AP)-based CL system. This technique depends on sandwich hybridization among MBs-labeled capture probes, target miRNAs and biotin-labeled reporter probes, conjugation of streptavidin-alkaline phosphatase(SA-AP) to biotin-labeled reporter probes, and CL detection of AP-linked targets. Detection of miR-21 with this technique demonstrated a high selectivity and an ultralow limit of detection(LOD) of 60 fM with an extraordinarily wide range of six orders of magnitudes. The quantitation could be achieved by direct detecting target miRNA in serum samples within a total time of 1.5 h and did not require reverse transcription and polymerase chain reaction(PCR)amplification. Therefore, this developed method shows great potential for early cancer diagnosis based on miRNAs as biomarkers. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level, and their aberrant expression occurs during the development of malignant diseases. Recently, miRNAs have been proposed as potential prognostic and predictive biomarkers for early diagnosis. However, , a major obstacle in rapid miRNA analysis from real samples is the lack of ultrasensitive and quantitative techniques. In this regard, the use of chemiluminescence (CL) system offers a highly sensitive strategy for detecting miRNAs. In this article, an ultrasensitive approach has been established for the quantification of miRNAs, using magnetic beads (MBs) and alkaline phosphatase (AP) -based CL system. This technique depends on sandwich hybridization among MBs-labeled capture probes, target miRNAs and biotin-labeled reporter probes, conjugation of streptavidin- alkaline phosphatase (SA-AP) to biotin-labeled reporter probes, and CL detection of AP-linked targets. Detection of miR-21 with th is technique demonstrated a high selectivity and an ultralow limit of detection (LOD) of 60 fM with an extraordinarily wide range of six orders of magnitudes. The quantitation could be achieved by direct detection target miRNA in serum samples within a total time of 1.5 h and did not require reverse transcription and polymerase chain reaction (PCR) amplification. Thus, this developed method shows great potential for early cancer diagnosis based on miRNAs as biomarkers.