Creating color difference and improving the color resolution in digital imaging is crucial for better application of color analysis. Herein, a novel color modulation analysis strategy was developed by using a homemade tunable multiband laser illumination device, in which the portions of R, G, and B components of the illumination light are discretionarily adjustable, and hence the sample color could be visually modulated continuously in the RGB color space. Through this strategy, the color appearance of single gold nanorods (AuNRs) under dark-field microscopy was migrated from the spectrally insensitive red region to the spectrally sensitive green-yellow region. Unlike the traditional continuous-wave light source illumination, wherein the small spectral variations in the samples within a narrow spectral range are averaged by the whole spectrum of the light source, leading to little color difference, the application of sharp, multiband laser illumination could enlarge the color separation between samples, thus resulting in high spectral sensitivity in color analysis. By comparing the corresponding color evolution processes of different samples as the multiband combination of the laser illumination was changed, more efficient color separation of AuNRs was achieved. With this instrument and single [email protected] as the sulfide probe, we achieved high throughput and highly sensitive detection of sulfide at a detection limit of 0.1 nM, a more than 2 orders of magnitude improvement compared to the previous color sensing scheme. This strategy could be utilized for nanoparticle identification, evaluation, and determination in biological imaging and biochemical analysis. © 2018 American Chemical Society.