Results of in situ fluorescence investigations on chlorophyll-a (chl-a) of phytoplankton in the Sea of Azov (May to June 2018) and in the Don River estuary (September to November 2019) are presented. Continuous data series of chl-a fluorescence were obtained with flow-through fluorometers. Discrete reference measurements of chl-a concentration and phytoplankton biomass were performed by standard methods for the sake of comparison and eventual corrections. The fluorescence intensity values measured in a lateral surface salinity gradient of the Sea of Azov were found correlating with the obtained data on the chl-a concentration (R2 = 0.88, n = 27) and phytoplankton biomass (R2 = 0.90, n = 11). Instead, there was a weak correlation (R2 = 0.40, n = 33) between the fluorescence and phytoplankton biomass found in the estuary. This disparity in correlations was explained by the difference in conditions during measurements, which affected the fluorescence. There were no significant changes in both temperature and coenotic composition of phytoplankton in the course of the marine expedition. The measurements on the river covered a period characterized by the seasonal variations in the phytoplankton composition and by noticeable temperature fluctuations.