Synthesis and self-assembly of stimuli-responsive amphiphilic alternating copolymers (AAC) are an emerging land of tremendous possibilities. Herein, by combining backbone polyethylene glycol (PEG) with pendent oligo-polyglycol simultaneously, two alternating LCST segments are knitted through enzymatic synthesis, giving a series of alternating poly[(PEG400-a-succinic acid)-co-(diol(3EG)-a-succinic acid)] (PPSDS) for the first time. All the PPSDSs show only one-step sharp temperature responsiveness in transmittance-temperature curve owing to stabilization effect of PEG400. The cloud points can be linearly controlled by simply adjusting the feeding ratio of PEG400/diol-3EG. Referring to published works and H-1-NMR spectra in D2O, all the obtained AAC formed penetrable nanovesicles under 4 degrees C. The TEM and H-1-NMR results confirmed that when heated to 18 degrees C, PPSDS of "9/1" transformed from nanovesicles to large-compound micelles due to large hydrophobic volume, while other PPSDS of "8/2, 7/3, 6/4" retained the vesicle structures, except that the hydrophilic layer turned from PEG400 + diol(3EG) to PEG400 alone, leading to the size reduction. The temperature-controlled size "expansion and contraction" of nanovesicles was unique for AAC, which was potentially good for enhancing loading rate. Further heating above cloud point resulted in the destruction of nanostructures and irregular intermolecular aggregations. The first reported dual temperature-responsive AAC was innovative in structure design, providing a potential opportunity for the design and synthesis of controllable self-assemble structures and smart biomacromolecules in biomedical applications.