We have implemented a hand-held photoacoustic and ultrasound probe for image-guided needle biopsy using a modified clinical ultrasound array system. Pulsed laser light was delivered via bifurcated optical fiber bundles integrated with the hand-held ultrasound probe. We photoacoustically guided needle insertion into rat sentinel lymph nodes (SLNs) following accumulation of indocyanine green (ICG). Strong photoacoustic image contrast of the needle was achieved. After intradermal injection of ICG in the left forepaw, deeply positioned SLNs (beneath 2-cm thick chicken breast) were easily indentified in vivo and in real time. Further, we confirmed ICG uptake in axillary lymph nodes with in vivo and ex vivo fluorescence imaging. These results demonstrate the clinical potential of this hand-held photoacoustic system for facile identification and needle biopsy of SLNs for cancer staging and metastasis detection in humans.