The development of H2O2-responsive fluorescent glycopolymers with high specificity, sensitivity, and water solubility is a promising strategy for developing probes to detect the production and delivery of endogenous H2O2. Herein, two series of fluorescent glycopolymers (boronic acid derivatives, PG-PFE, and borate derivatives, PG-PFW) were synthesized through esterification and the Williamson reaction, respectively. To increase the sensitivity of the fluorescent glycopolymers, the effect of the reaction conditions on the grafting ratio was investigated in detail. Compared with the PG-PFE samples, the PG-PFW samples had a higher grafting ratio due to the highly efficient Williamson reaction. The structure and properties of the fluorescent glycopolymers were characterized by using nuclear magnetic resonance spectroscopy, gel permeation chromatography, ultraviolet spectroscopy, and photoluminescent spectroscopy. The obtained results showed that all of these fluorescent glycopolymers had good water solubility, low cytotoxicity, and selective responsiveness toward H2O2. HPG-PFW-5, with the highest grafting ratio, displayed a higher sensitivity toward H2O2 and higher stability during hydrolysis than the other materials. In confocal laser scanning microscope images, all of the fluorescent glycopolymers were observed only in cellular mitochondria, and HPG-PFW-5 exhibited a high degree of overlap with Mitotracker Red because of its high sensitivity. Two series of fluorescent glycopolymers (boronic acid derivatives, PG-PFE, and borate derivatives, PG-PFW) were synthesized through different methods. Compared with the PG-PFE samples, the PG-PFW samples had a higher grafting ratio due to the highly efficient Williamson reaction. The fluorescent glycopolymers possessed good water solubility, low cytotoxicity and selective responsiveness toward H2O2. Meantime, the fluorescent glycopolymers were imaged only in cellular mitochondria based on the fact that endogenous H2O2 is mainly distributed in cellular mitochondria.