With propagating through a dispersive medium, the temporal–spectral profile of optical pulses should be inevitably modified. Although such dispersion effect has been well studied in classical optics, its effect on a single-photon wave-packet has not yet been entirely revealed. In this paper, we investigate the effect of dispersion on indistinguishability between single-photon wave-packets through the Hong–Ou–Mandel (HOM) interference. By dispersively manipulating two weak coherent single-photon wave-packets which are prepared by attenuating mode-locked laser pulses before interfering with each other, we observe that the difference of the second-order dispersion between two optical paths of the HOM interferometer can be mapped to the interference curve, indicating that (i) with the same amount of dispersion effect in both paths, the HOM interference curve must be only determined by the intrinsic indistinguishability between the wave-packets, i.e., dispersion cancellation due to the indistinguishability between Feynman paths; and (ii) unbalanced dispersion effect in two paths cannot be canceled and will broaden the interference curve thus providing a way to measure the second-order dispersion coefficient. Our results suggest a more comprehensive understanding of the single-photon wave-packet and pave ways to explore further applications of the HOM interference.