A single molecule detection technique was developed by the combination of a single channel poly (dimethylsiloxane)/glass micro-fluidic chip and fluorescence correlation spectroscopy (FCS). This method was successfully used to determine the proportion of two model components in the mixture containing fluorescein and the rhodamine-green succinimidyl ester. A single molecule detection technique was developed by the combination of a single channel poly (dimethylsiloxane) / glass micro-fluidic chip and fluorescence correlation spectroscopy (FCS). This method was successfully used to determine the proportion of two model components in the mixture containing fluorescein and the rhodamine-green succinimidyl ester.