Drug-loaded nanogels have attracted extensive research interest and shown great potential for many biomedical applications. A facile preparation method for drug-loaded nanogels is desirable to be developed. In this study, a range of nanogels were prepared by combining thiol-ene click reaction and photopolymerization at 532 nm for in situ loading 5-Fluorouracil (NG-S). The drug loading amount was improved by introducing mercaptosuccinic acid (MCA) to trigger thiol-ene click reaction. The drug loading capacity and swelling ratio characteristics of NG-S could be tuned by changing the proportion of PEGDA/MCA and the initial dose of drug. Especially, the drug release performance of the NG-S showed that 5-Fluorouracil presented the relatively excellent behavior of controlled release with a sustained release time up to 20 h in PBS (pH 7.4) at 37 °C. The fitting results of NG-S based on R 2 were consistent with the First order, Weibull and Biexponential kinetic models. The strategy through combination of thiol-ene click reaction & photopolymerization is firstly proposed to prepare nanogels to improve the drug loading amount of 5-Fluorouracil and tune its release. • Nanogels loading drug was prepared in situ by combining thiol-ene click reaction and photopolymerization at 532 nm. • Thiol-ene click reaction can tune the crosslinking density and polymerization structure of nanogels to improve the drug loading amount. • Drug loaded nanogels have excellent sustained-release behavior.