As one of the toxic by-product of aerobic metabolism, hydrogen peroxide (H2O2), at uncontrolled levels and distributions, is a sign of oxidative stress, aging and disease. However, H2O2 also plays an essential part in normal physiological system. H2O2 levels are regulated by many enzymes and metabolites that generate or break-down H2O2. In macrophages, the presence of invading pathogens activates the production of microbicidal levels of H2O2 by NADPH oxidase (Nox). Isoforms of Nox are expressed in many non-phagocytic cells and tissues. Nox-generated H2O2 is a secondary messenger involved in signaling for growth, proliferation, differentiation and controlled cell death; these variations in downstream biological effects are regulated by both the spatial and temporal production of H2O2. Small molecule fluorescent probes bearing boronate ester moieties have been developed for chemoselective detection of H2O2 in both oxidative stress levels and cellular signaling events. This dissertation describes the design, synthesis, characterization and application of new boronate-based fluorescent probes with added functionality. Peroxy-Lucifer-1 (PL1) and Peroxy-Naphthalene-1 (PN1) are ratiometric fluorescent probes that can detect oxidative bursts in immune response events. Ratiometric probes allow simultaneous detection of two signals from the reacted and unreacted probes in the same sample, providing a built-in correction for variations such as uneven probe loading, sample environment and detection efficiency. PN1 also has a high two-photon cross section. The increased penetration depth of near-infrared excitation light allows the detection of H2O2 in tissue specimens with PN1. SNAP-Peroxy-Green-1 (SPG1) and SNAP-Peroxy-Green-2 (SPG2) are capable of detecting local concentration of H2O2 in subcellular compartments such as mitochondria, endoplasmic reticulum, nucleus, and plasma membrane. The precise localization of probes to the targeted organelle is facilitated by highly specific recognition of the SNAP ligand bound to the probe by the SNAP fusion protein. Furthermore, simultaneous detection of H2O2 at two different locations is feasible by using a SNAP tag with an orthogonal CLIP tag; such combined use of SNAP and CLIP tags is assisted by the expanding color palette of SNAP and CLIP peroxy probes. Multi-modal probes using PAMAM-G5 dendrimer platform was developed for the real-time imaging of the interplay between H2O2 and other physiological events. Coordination of the oxidative burst and progressive acidification in phagosomes of macrophages was elucidated with G5-SNARF2-PF1-Ac, a nanoprobe decorated with the H2O2 sensing module PF1 and pH sensor SNARF2.