Herein, a renewed prominence towards the synthesis of poly(alkylene citrate) (PAC) biomaterials and their detailed chemical, structural and mechanical characterization has been reported. Based on the modifications to the PAC synthesis protocol introduced in this study, the fabrication process was significantly streamlined, the reaction yields were increased, and the homogeneity of the final materials was found to be substantially improved. Comprehensive nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) studies of the fabricated prepolymers shed light on the mechanism of the PAC cross-linking process and supported the design of materials with enhanced biocompatibility. Therefore, the initial molar ratio of the reagents involved in the synthesis of PAC materials was found to be pivotal to both the biological and mechanical properties of the final products. Moreover, cell viability and proliferation assays revealed enhanced biocompatibility of the materials formulated with a molar ratio of diol over citric acid (3 : 2 mol/mol) in comparison to the most commonly described 1 : 1 analogue without affecting the possibility of further functionalization. Furthermore, this work creates a new paradigm for prospective studies on the properties of modified PAC materials and their application in medicine and tissue engineering.