Local drug injection therapy for tumor site, as a neoadjuvant chemotherapy method, shows important significance in clinical application; however, it obtains unsatisfying therapeutic effect due to the serious toxic and side effect in normal tissues caused by drug diffusion or complexity of the preparation. In this article, the influence factors of the gelling time of traditional Chitosan (CTS) thermo-sensitive hydrogels were analyzed, and the gelling properties were improved significantly, and a thermosensitive hydrogel with precisely regulated gelling time was obtained through a green and simple preparation method, and the shortest gelling time (gelling time = 27 ± 2 s) of this hydrogel was 5% of that of the common CTS thermo-sensitive hydrogels. After loaded with different chemotherapy drugs with different pH values (gemctiabin hydrochloride, levofloxacin, and 5-foluorouracil), the hydrogels' gelling performance was not affected, while the gelling time could be shortened by 5-foluorouracil, effectively hindering the drug loss at the early stage of sustained release. in vitro and in vivo experiments proved that precise encapsulation toward tumors with different volumes was achieved by the hydrogels, with minimal damage to surrounding normal tissues and higher utilization of drugs in tumor sites, ultimately achieving better tumor therapeutic effect. In conclusion, the new thermo-sensitive hydrogels with precisely regulated gelling time showed great significance and potential for drug delivery and neoadjuvant chemotherapy. K E Y W O R D S drug-controlled release, gelling time, neoadjuvant chemotherapy, therapy in situ, thermosensitive hydrogels 1 | INTRODUCTION Thermo-sensitive hydrogels could achieve transformation between liquid and hydrogels according to the actual environmental temperature and act as a drug reservoir in injection site, with the advantages of controlled release, targeted drug delivery, and low toxicity (Culver,