New biodegradable amphiphilic ABA triblock copolymers consisting of poly(ethylene oxide) (PEO) as the hydrophilic block and poly [(R)-3-hydroxy butyrate] (PHB) as the hydrophobic block were synthesized by coupling two chains of methoxy-PEO-monocarboxylic acid with a low molecular weight telechelic hydroxylated PHB (PHB-diol) chain in the middle. The PHB-diol was prepared from high molecular weight natural source PHB by transesterification reaction with diethylene glycol. The target triblock copolymers were isolated and purified from the reaction mixtures through repeated precipitation and fractionation. The structures and molecular characteristics of the PEO-PHB-PEO triblock copolymers were studied by GPC, H-1 NMR, and FT-IR. All of the triblock copolymers had unimodal peaks in the GPC chromatographs. The chain architecture of the triblock copolymers was confirmed by the 1H NMR spectra. A combination of GPC and H-1 NMR analysis determined the lengths of the PEO and PHB blocks of the copolymers. The thermal properties, phase separation, and crystalline characteristics of the copolymers were studied using thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and wide-angle X-ray diffraction (XRD). TGA analysis showed that the triblock copolymers undergo thermal degradation in two separate steps for the PHB and PEO blocks, from which the PHB contents were calculated and compared with those determined from GPC and 1H NMR. The TGA results also showed that the triblock copolymers have better thermal stability than their respective precursors. Both DSC and XRD analysis showed that separate crystalline phases are formed by PEO and PHB blocks in the copolymers, except for those with a very short PHB block length. The crystallinity of PHB block in the copolymers increases as compared with the pure PHB precursor, presumably because of the presence of the soft PEO block. In contrast, the crystallinity of the PEO block in the copolymers decreases as compared with the PEO precursor because of the presence of the hard PHB block, which restricts the crystallization of the PEO blocks.