The effect of flexible spacer length on the liquid crystalline property of ABA-type triblock copolymers containing azobenzene groups was investigated. For the study, the monomers, n-[4-(4-ethoxyphenylazo)phenoxy]alkyl methacrylates with varying methylene groups (n = 0, 2, 6) were used to synthesize a series of azobenzene-containing amphiphilic triblock copolymers PAnC-PEG-PAnC by atom transfer radical polymerization (ATRP). Differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and one-dimensional X-ray diffraction (1D WAXD) have shown that the glass transition temperatures of these copolymers decreased with increasing n, PA0C-PEG-PA0C has no mesophase, while both PA2C-PEG-PA2C and PA6C-PEGPA6C have a nematic mesophase. These differences derive from the length of spacer groups between the polymer backbone and side-chain LC monomers.