The invention discloses a calibration method for a fluorescence method chlorophyll a online monitor. The calibration method comprises: preparing a plurality of pure algae variety samples having four different types; respectively determining the chlorophyll a values of different pure algae variety samples through a traditional laboratory detection method and an online monitor, and respectively calculating the ratios between the chlorophyll a values obtained through the different methods so as to be used as calibration coefficients; carrying out algae enrichment on the detected samples, and determining a dominant algae variety; starting the automatic cleaning function of the online monitor; regulating the light transmittance of pure water of the online monitor at more than 99%; determining an actual water sample through the online monitor to obtain the chlorophyll a components of different algae varieties and the total chlorophyll a; and calibrating the detected data of the corresponding superiority category in the determined value of the online monitor by using the calibration coefficients, and re-calculating the online detection value so as to obtain the final chlorophyll a detection value. With the calibration method of the present invention, the data accuracy of the fluorescence method chlorophyll a online monitor can be significantly improved.