Microcystin with leucine and arginine (MCLR) is a hepatotoxic toxin produced by cyanobacteria present in watercourses that causes cell death and tissue necrosis at high exposure levels. In this study, a sensitive non-competitive, Open sandwich Enzyme-linked Immunosorbent Assay (OS-ELISA) with wide detectable range was developed for the quantification of MCLR. OS-ELISA is based on the principle of antigen-driving interaction enhancement between variable regions of an antibody. The genes for the variable regions of antibody 3A8 against MCLR were used to construct the phage display vector pDong1/Fab(3A8) to prepare assay elements. The OS-ELISA performed with phage-displayed antibody VH fragment and secreted light chain showed a limit of detection (LOD) of 0.14 nM and a wide working range from 0.14 to 10,000 nM MCLR. Another format of OS-ELISA that used purified Maltose Binding Protein-fused VL and VH-phage showed a lower LOD of 85 pM MCLR. Using the developed assay, the recovery rates for samples of lake or river water spiked with MCLR were found to range from 95.5% to 116.4%, suggesting that the assay has good potential to be used as a monitoring tool for MCLR in nature.