Combination of chemotherapy and photothermal therapy has been proposed for better treatment of metastatic colon cancer. In this study SN38, a highly potent cytotoxic agent, was conjugated to negatively charged hyaluronic acid (HA), which was deposited on the surface of the positively charged gold nanoparticles via electrostatic interaction. The drug conjugation and its interaction with gold nanoparticles were verified by 1H NMR and UV–vis spectroscopies, respectively. The prepared SN38-HA gold NPs are negatively charged spherical nanoparticles with an average size of 75 ± 10 nm. In vitro release study revealed that drug release in acidic conditions (pH 5.2) was faster than that in physiological pH. Red light emitting diode (LED, 630 nm, 30 mW) was used as a light source for photothermal experiments. The drug release in acidic conditions was increased up to 30% using red LED illumination (6 min) in comparison with experiment carried out indark. The cytotoxicity study on MUC1 positive HT29, SW480 colon cancer cells and MUC1 negative CHO cells, showed higher toxicity of the nanoparticles on HT29 and SW480 cell lines compared to CHO cells. Confocal microscopy images along with flow cytometry analysis confirm the cytotoxicity results. The incubation time for reaching IC50 decreases from 48 h to 24 h by LED illumination after nanoparticle treatment. Migratory potential of the HT29 and SW480 cell lines was reduced by co-application of SN38-HA gold NPs and LED radiation. Also anti-proliferative study indicates that LED radiation has increased the cytotoxicity of the nanoparticles and this effect is remained up to 8 days.