Fluorescence imaging in the second near-infrared window (NIR-II) has been an emerging technique in diverse in vivo applications with high sensitivity/resolution and deep tissue penetration. To date, the design principle of the reported NIR-II organic fluorophores has heavily relied on benzo[1,2-c:4,5-c’]bis([1,2,5]thiadiazole) (BBTD) as a strong electron acceptor. Here, we report the rational design and synthesis of a NIR-II fluorescent molecule with the rarely used [1,2,5]thiadiazolo[3,4-f]benzotriazole (TBZ) core to replace BBTD as the electron acceptor. Thanks to the weaker electron deficiency of the TBZ core than BBTD, the newly yielded NIR-II molecule (BTB) based nanoparticles have a higher mass extinction coefficient and quantum yield in water. In contrast, the nanoparticle suspension of its counterpart with BBTD as the core is nearly nonemissive. The NIR-II BTB nanoparticles allow video-rate fluorescence imaging for vasculature imaging in ears, hindlimbs, and the brain of the mouse. Additionally, its large absorptivity in the NIR-I region also promotes bioimaging using photoacoustic microscopy (PAM) and tomography (PAT). Upon surface conjugation with the Arg-Gly-Asp (RGD) peptide, the functionalized nanoparticles ensured targeted detection of integrin-overexpressed tumors through both imaging modalities in two- and three-dimensional views. Thus, our approach to engineering acceptors of organic fluorophores offers a promising molecular design strategy to afford new NIR-II fluorophores for versatile biomedical imaging applications.