MiRNAs are expressed in a tissue-specific fashion in the eyes and changes in miRNAs levels in aqueous humor (AH) may reflect the function of the eye and eye disease. Due to the low concentration of total miRNA in human aqueous humor, high volume of sample is required for RNA extraction prior to routine quantification such as RT-qPCR. However, limited volume of AH could be collected through surgery because of the characteristic of the eye. In addition, inefficiency of RNA-extraction kits could affect target miRNA quantification dramatically. AH-direct miRFLP assay was developed for quantification of target miRNAs in human aqueous humor samples. For the first time, accurate miRNA quantification in human AH was achieved with microliter scale sample loading. Higher copy numbers of target miRNAs were obtained in direct detection than in RNA-extraction solution. It indicates that AH-direct miRFLP assay was able to quantify target miRNAs more accurately with no requirement for RNA-extraction to avoid sampling variability.