The interaction between triblock copolymer Pluronic P123 and anionic surfactant Aerosol OT (AOT) has been studied using dynamic light scattering (DLS) and small angle neutron scattering (SANS). The copolymer/surfactant system has been investigated at a constant copolymer concentration of 0.5 wt% and with varying surfactant concentration up to ~5 mM. DLS results reveal that addition of AOT suppresses the micellization of P123. With the addition of AOT, P123–AOT mixed micelles are formed via hydrophobic association of the surfactant with the block copolymer micelles leading to the development of negative surface charge on copolymer micelles. The solubilization of the hydrocarbon chains of the surfactant, AOT, into the polypropylene oxide core of the block-copolymer micelle leads to increased electrostatic repulsion between the P123–AOT mixed micelles. SANS studies show a steady decrease in the core size and aggregation number of the micelles with the increasing concentration of AOT, consistent with the DLS results. The binding ability of AOT to the anticancer drug doxorubicin hydrochloride (DOX) was confirmed by dynamic surface tension measurements. The in vitro cytotoxicity studies in various cancer cell lines revealed that anticancer activity of the drug is not affected by complexation with AOT and DOX-loaded micelles are more potent to MCF-7 human breast cancer cells compared to other cell lines. The present findings can form a basis to design block copolymer based micellar carriers for drug delivery.