Background: Positron emission tomography (PET) images are being applied for defining radiotherapy targets. However, a recognized method for defining radiotherapy targets is lacking. We investigate the threshold to outline the radiotherapy target of a tumor on PET images and its influencing factors, and then expressed it by formula. Methods: PET imaging for spherical tumors with a different tumor diameter (D), under different system resolutions [full width at half maximum (FWHM)], in different backgrounds with different pixel sizes, was simulated. PET images were analyzed to determine the relationship between the threshold and the factors mentioned above. Finally, the simulation results were verified by phantom experiments. Results: The threshold decreased sharply with D for D < 2 FWHM, reached the minimum of 31% at D = 2 FWHM and then increased slowly, and it tended to constant for D > 8 FWHM. The threshold decreased with FWHM for FWHM < D/2, reached a minimum at FWHM = D/2, and then increased. The threshold increased with pixel size for D ≤ FWHM and decreased for D > FWHM. The threshold was independent of the background. The relationship between the threshold and its influencing factors was expressed as a formula. The results of the phantom verification indicated that the error of the target volume delineation that was calculated by the formula was less than 9%. Conclusions: The threshold changes with tumor size, resolution of the PET system and pixel size according to certain rules. The formula to calculate the threshold could provide a method to estimate threshold to outline the radiotherapy target (tumor).