Cells employ proteins to perform metabolic functions and maintain active physiological state through charge transfer and energy conversion. These processes are carried out in a narrow space precisely and rapidly

which

no doubt

bring great difficulty for their detection and dissection. Fortunately

in recent years

the development and expansion of single-molecule technique in protein research make monitoring the dynamical changes of protein at single-molecule level a reality

which also provides a powerful tool for the further exploration of new phenomena and new mechanisms of life activities. This paper aims to summarize the working principle and essential achievements of single-molecule technique in protein research in recent five years. We focus on not only dissecting the difference of nanopores

atomic force microscope

scanning tunneling microscope

and optical tweezers technique

but also discussing the great significance of these single-molecule techniques in investigating intramolecular and intermolecular interactions

electron transport

and conformational changes. Finally

the opportunities and challenges of the single-molecule technique in protein research are discussed

which provide a new door for single-molecule protein research.