Graphite-oxide (GO) nanofluids with enhanced thermal conductivity are successfully prepared using pulsed laser ablation in liquid (PLAL) of a graphite target in deionized water. The effects of laser frequencies are investigated on the variations of GO nanoparticles. The morphology, structure and composition of the nanoparticles are characterized using various spectroscopic techniques. During the PLAL process, graphite is oxidized to GO which is inherently hydrophilic, no surfactant is required in the preparation of nanofluid. The laser frequency significantly affects the size and morphology of the GO nanoparticles during laser ablation, leading to a profound variation in the thermophysical properties of the GO nanofluids. At the laser frequency of 10 Hz, the maximum thermal conductivity enhancement of 82% is achieved at a temperature of 50 °C while the maximum viscosity increment is recorded at a temperature of 30 °C. This study shows the great potential of the PLAL method in synthesizing GO nanofluid with anomalously enhanced thermal conductivity.