Developing　chemiluminescence　probe　with　a　slow　kinetic　profile,　even　a　constant　emission　within　analytical　time,　would　improve　the　analytical　sensitivity,　but　still　remains　challenging.　This　work　reports　a　novel　strategy　to　afford　long-lasting　in　vivo　imaging　by　developing　a　self-assembled　chemiluminophore　HPQCL-Cl　via　the　introduction　of　the　hydrogen-bond-driven　self-assembled　dye　HPQ　to　Schaap＇s　dioxetane.　Compared　with　classical　chemiluminophore　HCL,　self-assembled　HPQCL-Cl　was　isolated　from　the　physiological　environment,　thereby　lowering　its　deprotonation　and　prolonging　its　half-life.　Based　on　HPQCL-Cl,　the　long-lasting　in　vivo　imaging　of　9L-lacz　tumor　was　achieved　by　developing　a　β-gal-responsive　probe.　Its　signals　remained　constant　(＜5%　change)　for　about　20　min,　which　may　provide　a　wide　time　window　for　the　determination　of　β-gal.　This　probe　also　showed　high　tumor-to-normal　tissue　ratio　throughout　tumor　resection,　highlighting　its　potential　in　image-guided　clinical　surgery.　©　2022