Since the serendipitous discovery of heavy-chain antibodies in Camelidae 20 years ago, the smallest single-domain antigen-binding fragment, known as VHH or nanobody, has received growing attention. In comparison with traditional antibodies, VHHs performs equally high specificity and affinity and low toxicity to targets, and has the ability to inhibit the formation of enzymes and enter the receptor gap. Hence, VHHs has been recently regarded as the highly valued protein and applied in multiple fields, including fundamental research, diagnosis, therapeutics, food science, etc. Today, based on the past achievements, an increasing number of academic and industrial groups begin to explore innovative VHH applications. In this review, we make a thorough retrospect of the unique features and explore novel implementations of VHHs in diverse forms in different fields. In addition, we also propose the potential future issues and problems of VHHs in many fields.