The precise modulation of the π-conjugated bridge within a probe is of considerable importance for the development of reaction-based fluorescent probes endowed with specific functionalities. Here, a series of naphthalimide-based fluorescent probes with a fluorescence ratio and attenuated sensing properties for H2O2 have been designed by precisely tuning the type of the π-conjugated bridge located between the boric acid recognizing the moiety and the naphthalimide fluorophore. The modulation of the π-conjugated bridge species mainly focused on three structural units, thiophene, benzene, and furan, among which the thiophene helped us to construct the most efficient naphthalimide-based fluorescent probe (E)-(5-(((2-(2-mercaptoethyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl)imino)methyl)thiophen-2-yl)boronic acid (MOHB-IMTP). It demonstrated the most remarkable fluorescence changes before and after the reaction and thus a significantly superior sensing performance for H2O2, including fairly good selectivity and a lower limit of detection (LOD, 38.5 nM). In addition, based on cellulose-induced aggregation of the reaction products, the MOHB-IMTP/cellulose probe was further developed and proven to have a faster and more sensitive (4.0 nM) detection toward H2O2. We postulate that this π-conjugated bridge modulation and cellulose signal enrichment strategy will actively promote the development of high-performance fluorescent probes and sensing devices.