We synthesized sustainable 2,4-di-tert-butylphenoxymethyl-substituted polystyrenes (PDtBP#, # = 88, 68, 35, and 19, where # is molar percent contents of 2,4-di-tert-butylphenoxymethyl moiety), using post-polymerization modification reactions in order to study their liquid crystal (LC) alignment behaviors. In general, LC cells fabricated using polymer film with higher molar content of 2,4-di-tert-butylphenoxymethyl side groups showed vertical LC alignment behavior. LC alignment behavior in LC cell was related to the surface energy of the polymer alignment layer. For example, when the total surface energy value of the polymer layer was smaller than about 29.4 mJ/m2, vertical alignment behaviors were observed, generated by the nonpolar 2,4-di-tert-butylphenoxymethyl moiety with long and bulky carbon groups. Orientation stability was observed at 200 °C in the LC cells fabricated using PDtBP88 as the LC alignment layer. Therefore, as a natural compound modified polymer, PDtBP# can be used as a candidate LC alignment layer for environmentally friendly applications.