Fluorescence imaging is a well-established optical modality that has been used to localize and track fluorophores and has demonstrated great potential for surgical guidance. Despite the variety of fluorophores currently being researched, many existing intraoperative fluorescence imaging systems are specifically designed for a limited number of applications. We present a modular wide-field fluorescence overlay tissue imaging system for intraoperative surgical guidance that is comprised of commercially available standardized components. Its modular layout allows for the accommodation of a broad range of fluorophores, fields of view (FOV), and spatial resolutions while maintaining an integrated portable design for intraoperative use. Measurements are automatic and feature a real-time projection overlay technique that intuitively displays fluorescence maps directly onto a [Formula: see text] FOV from a working distance of 35 cm. At a 20-ms exposure time, [Formula: see text] samples of indocyanine green could be measured with high signal-to-noise ratio and was later tested in an mouse model before finally being demonstrated for intraoperative autofluorescence imaging of human soft tissue sarcoma margins. The system's modular design and ability to enable naked-eye visualization of wide-field fluorescence allow for the flexibility to adapt to numerous clinical applications and can potentially extend the adoption of fluorescence imaging for intraoperative use.