Compartmentalization and unidirectional cross-domain molecule shuttling are omnipresent in proteins, and play key roles in molecular recognition, enzymatic reaction, and other living functions. Nanomachinery design emulating these biological functions is being considered as one of the most ambitious and challenging tasks in modern chemistry and nanoscience. Here, we present a biomimetic nanomachinery design using single-chain technology. Stepwise complex of the outer blocks of water-soluble linear ABC triblock terpolymer to copper ions yields dumbbell-shaped single-chain nanoparticle. A novel nanomachine capable of compartmentalization and unidirectional cross-domain molecule shuttling has been achieved upon ascorbic acid reduction, leading to synergistically donating/accepting copper centers between discrete double heads, overall dumbbell-to-tadpole configurational transition, and intake of oxidized ascorbic acid into reconstructed head. Subsequent air oxidation results in the inverse molecule shuttling and configurational transition processes. This is the first demonstration of biomimetic nanomachinery design that is capable of compartmentalization and unidirectional cross-domain molecule shuttling, exemplified simply using a new single-chain technology.