We prepared nanoparticles by a temperature-induced phase transition in a mixture of Pluronic F-68 and liquid PEG (polyethylene glycol, molecular weight: 400) containing paclitaxel (PTX) with a fast, simple, continuous and solvent-free process. The liquid PEG is used as solubilizer of PTX and the polymer for the encapsulation of PTX is composed of Pluronic F-68. At the phase transition temperature, the polymer mixture was changed to the liquid phase, and stirring the liquid 0 °C to form Pluronic nanoparticles. The morphology and size distribution of the prepared Pluronic nanoparticles were observed using FE-SEM and TEM, and a particle size analyzer and cryo-TEM were used to observe the shape of paclitaxel-loaded Pluronic nanoparticles in an aqueous state. To apply Pluronic nanoparticles as a delivery system for cancer therapy, the release pattern of PTX, a model anti-cancer drug, was observed and the tumor growth was monitored by injecting the PTX-loaded Pluronic nanoparticles into the tail veins of tumor-bearing mice. We also evaluated the time-dependent excretion profile, in vivo biodistribution, circulation time, and tumor targeting ability of PTX-loaded Pluronic nanoparticles using non-invasive live animal imaging technology. In the early stage within 7h of release, the loaded PTX was rapidly released and the sustained release was observed for up to 48 h. In vivo studies, PTX-loaded Pluronic nanoparticles were observed with higher anti-tumor efficacy compared with PTX formulated in Cremophor EL.