Purpose Photo-gamma fusion lymphoscintigraphy (PGFLS) was developed by overlying a conventional planar gamma image on a photograph for the guidance of sentinel node biopsy. The feasibility and accuracy of PGFLS was assessed in breast cancer patients. Methods A digital camera and a gamma camera were coordinated to obtain photograph and gamma images from the same angle. Using the distance to the object and calibration acquisition with a flat phantom and radioactive markers, PGFLS was performed both in phantom and in patients without fiducial markers. Marker-free PGFLS was verified using flat phantom, anthropomorphic phantom with markers simulating sentinel nodes and breast cancer patients. In addition, the depth of the radioactive marker or sentinel node was calculated using two gamma images taken at right angles. The feasibility and accuracy of PGFLS were assessed in terms of mismatch errors of co-registration and depth with reference to the data from SPECT/CT.