Fluorescence imaging is considered as one of the most powerful techniques for monitoring biomolecule activities in living systems. Near-infrared (NIR) light is advantageous for minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have developed a new NIR fluorescent dye, namely, RB-1, based on the Rhodamine B scaffold. RB-1 exhibits excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, and high photostability. In particular, RB-1 displays both absorption and emission in the NIR region of the "biological window" (650-900 nm) for imaging in biological samples. RB-1 shows absorption maximum at 614 nm (500-725 nm) and emission maximum at 712 nm (650-825 nm) in ethanol, which is superior to those of traditional rhodamine B in the selected spectral region. Furthermore, applications of RB-1 for fluorescence imaging in living cells and small animals were investigated using confocal fluorescence microscopy and in vivo imaging system with a high signal-to-noise ratio (SNR = 10.1).