A novel assay detection method for microcystin-LR (MC-LR) in surface water is researched and established which combines surface plasmon resonance (SPR) technique with immunoassay method. SpreetaTM sensor is adopted in the SPR flow injection analysis (FIA) system. The reusable biosensor chip is functionalized with (MC-LR)-BSA using covalent-coupling method. Experimental studies of SPR immunoassay method for MC-LR are carried out, the results show that relative standard deviation (RSD), quantitative range, detective limit and half maximal inhibitory concentration (CI50) are 1.0% (n=6), 2~32 ng/mL, 0.63 ng/mL and 10.7 ng/mL,respectively. Recovery rate of blank sample and different water samples spiked MC-LR ranges from 90% to 113%. It is concluded that tap water does not contain MC-LR while pond water contains 2.46 ng/mL MC-LR in real sample assay. The experimental results demonstrate that the SPR immunoassay can well meet drinking water guidelines of the World Health Organization (WHO) for MC-LR.