Precise photon flux measurement of single photon sources (SPSs) is essential to the successful application of SPSs. In this work, a novel method, to our knowledge, was proposed for direct measurement of the absolute photon flux of single photon sources with a femtosecond laser multiphoton microscope. A secondary 2-mm-diameter aperture was installed under the microscope objective to define the numerical aperture (NA) of the microscope. The defined NA was precisely measured to be 0.447. An LED-based miniaturized integrating sphere light source (LED-ISLS) was used as a standard radiance source to calibrate the photon flux responsivity of the multiphoton microscope, with the defined NA. The combined standard uncertainty of the measured photon flux responsivity was 1.97%. Absolute photon flux from a quantum-dot based emitter was measured by the multiphoton microscope. The uncertainty of the photon flux was evaluated to be 2.1%. This work offers a new, to our knowledge, radiometric method for fast calibration of photon flux responsivity of microscopes, and absolute photon flux calibration of single photon sources.