The invention relates to a gold nanosphere/melanin composite nanometer opto-acoustic probe with ultra-strong opto-acoustic effect and preparation thereof. The gold nanosphere/melanin composite nanometer opto-acoustic probe is prepared by compounding dodecylamine-modified gold nanospheres with melanin nanometer particles coating the dodecylamine-modified gold nanospheres, wherein a mass ratio of the dodecylamine-modified gold nanospheres to the melanin nanometer particles is 0.1-10: 1. Compared with the prior art, the composite nanometer opto-acoustic probe provided by the invention can realize circulation in the body for a certain period of time and can be disposed at a focus part after completion of opto-acoustic imaging; and the dissociated small-sized gold nanospheres can be removed via metabolism of the kidney and are thus prevented from accumulation in biological tissue and incurrence of damage.