Polydiacetylene (PDA) is an attractive material due to its unique chromatic transition properties by external stimuli. Its blue-to-red color transitions observed by the naked eye make the PDA a promising optical biosensor. However, the chromatic transitions of most PDA materials are irreversible, leading to restricted applications of PDA-based biosensors. In this study, we suggest that the colorimetric reversibility of PDA by the thermal-photo stimuli cycles is closely related to the difference in hydrogen bond strength and ionic interactions between adjacent headgroups of PDA vesicles. By fabricating three types of PDA vesicles consisting of single or complex headgroups, their reversibility in color change was compared. The PDA vesicles composed of both carboxyl- and amine-terminated monomers (poly(PCDA/PCDA-EDA)) at a molar ratio of 1:1 exhibited noticeable chromatic reversibility with colorimetric response (CR) values of 84–91% and 24–61% during heating and UV irradiation cycles, respectively. And, the reversibility of the poly(PCDA/PCDA-EDA) vesicles under different pH conditions was examined to assess the effect of ionic interactions. To investigate the changes in the backbone and strength of hydrogen bonds in the prepared PDA vesicles, Raman and Fourier transform infrared (FT-IR) spectroscopies were executed, respectively. These results demonstrate that the hydrogen bond strength and ionic interactions of PDA vesicles with different terminal functional groups plays an important role in "red to blue" backward transitions, which can ultimately be applied to the development of recyclable PDA materials. [Display omitted] • A methodical approach to manipulate the colorimetric reversibility of polydiacetylene (PDA) is presented. • Three types of PDA vesicles consisting of single or complex headgroups are fabricated. • Poly(PCDA/PCDA-EDA) with complex headgroups exhibits distinct chromatic reversibility under thermal-photo stimuli cycles. • The PDA with stronger bonding force between headgroups shows reversible color transitions triggered by UV irradiation. • Structural alterations of PDA vesicles are intimately connected to their color response. [ABSTRACT FROM AUTHOR]