Environment-responsive fluorophores with aggregation-caused quenching (ACQ) properties have been applied to track nanocarriers with reduced artefacts caused by unbound or free fluorophores but suffer from incomplete fluorescence quenching and significant re-illumination, which undermine bioimaging accuracy. Herein, through structural modifications to reinforce the hydrophobicity, planarity and rigidity of fluorophores with an aza-BODIPY framework, probes featuring absolute ACQ (aACQ) and negligible re-illumination are developed and evaluated in various nanocarriers. aACQ probes, FD-B21 and FD-C7, exhibit near-infrared emission, high quantum yield, photostability, water sensitivity, and negligible re-illumination in blood, plasma and 1% Tween-80 in contrast to ACQ probe P2 and conventional probe DiR. All nanocarriers can be labeled efficiently by the tested fluorophores. Polymeric micelles (PMs) labeled by different aACQ probes manifest similar biodistribution patterns, which however differ from that of DiR-labeled PMs and could be ascribed to the appreciable re-illumination of DiR. Significantly lower re-illumination is also found in aACQ probes (2%–3%) than DiR (20%–40%) in Caco-2, Hela, and Raw264.7 cells. Molecular dynamics simulations unravel the molecular mechanisms behind aggregation and re-illumination, supporting the hypothesis of planarity dependency. It is concluded that aACQ fluorophores demonstrate excellent water sensitivity and negligible fluorescence re-illumination, making themselves useful tools for more accurate bioimaging of nanocarriers.