The effects of pH and Cu2+ on the detection of the Microcystin-LR based on evanescent wave all-fiber immunosensor (EWAI) and their elimination were studied. The experimental results indicated that both low pH and high pH greatly affected the immunoassay results of MC-LR. When pH was less than 6 or higher than 8, the fluorescence signals detected by immunosensor system obviously reduced with the decrease or increase of pH. While pH ranged from 6 to 8, IC50 and the linear working range of MC-LR calculated from the calibration curve were 1.01-1.04 microg/L and 0.12-10.5 microg/L, respectively, which was favourable for MC-LR immunoassay. Low concentration Cu2+ rarely affected the detection performance of MC-LR. When the concentration of CuSO4 was higher than 5 mg/L, the fluorescence signal detected by EWAI clearly decreased. Furthermore, when the concentration of CuSO4 equalled to 10 mg/L, the fluorescence signal detected reduced 70%. The influence of Cu2+ on the immunoassay could effectively be compromised when 1% EDTA was added to the pre-reaction mixture.