Understanding the control of the optical and plasmonic properties of unique nanosystems--gold nanostars--both experimentally and theoretically permits superior design and fabrication for biomedical applications. Here, we present a new, surfactant-free synthesis method of biocompatible gold nanostars with adjustable geometry such that the plasmon band can be tuned into the near-infrared region 'tissue diagnostic window', which is most suitable for in vivo imaging. Theoretical modelling was performed for multiple-branched 3D nanostars and yielded absorption spectra in good agreement with experimental results. The plasmon band shift was attributed to variations in branch aspect ratio, and the plasmon band intensifies with increasing branch number, branch length, and overall star size. Nanostars showed an extremely strong two-photon photoluminescence (TPL) process. The TPL imaging of wheat-germ agglutinin (WGA) functionalized nanostars on BT549 breast cancer cells and of PEGylated nanostars circulating in the vasculature, examined through a dorsal window chamber in vivo in laboratory mouse studies, demonstrated that gold nanostars can serve as an efficient contrast agent for biological imaging applications.