The invention discloses a small molecule probe based on nano-gold and aptamer and a preparation method of the small molecule probe. The small molecule probe comprises nano-gold particles, more than one type of sulfydryl modifying deoxyribonucleic acid (DNA) fragment assembled on the nano-gold particles and more than one type of aptamer which is marked by fluorescent groups, and at least a part of fragments of the more than one type of the aptamer are complemented and hybridized with the more than one type of sulfydryl modifying DNA group. The small molecule probe is short in detecting time, simple in operation, high in specificity, good in repeatability, high in accuracy, and the false positive result is effectively reduced. By means of the aptamer technology, a high detection sensibility is achieved, and various molecules can be simultaneously detected.