Bilirubin is an insoluble yellow pigment produced from heme catabolism and serves as a diagnostic marker of liver and blood disorders. Here, a systematic study of several interactions and arrangements between different forms of natural bilirubin and poly-5, 2[Formula: see text]-5[Formula: see text], 2[Formula: see text]-terthiophene-3-carboxylic acid/Mn(II)2complex, PTTCA–Mn(II)2, as a biosensor of bilirubin has been investigated extensively. The PTTCA–Mn(II)2biosensor detects natural bilirubin through the mediated electron transfer by the Mn[Formula: see text]. Initially, density functional theory (DFT) using B3LYP and different basis sets including 6-31G* and 6-311G** has been employed to calculate the details of electronic structure and electronic energies of natural biliverdin and [Formula: see text]-, [Formula: see text]- and [Formula: see text]-bilirubin. Next, the interaction of the PTTCA–Mn(II)2biosensor, being in three possible spin states, with [Formula: see text]-, [Formula: see text]- and [Formula: see text]-natural bilirubin with 1:1 and 1:2 stoichiometry using UB3LYP/6-31G* method has been investigated. Natural population analysis (NPA) calculations have been used to derive more suitable interaction sites of bilirubin with Mn[Formula: see text] ions in PTTCA–Mn(II)2biosensor. Investigation of different manganese complexes with bilirubin shows that the most stable complex is high spin state (total electron spin [Formula: see text]) rather than intermediate and low spin states with 1:2 stoichiometry. Also, the temperature effect and interferences from other biological compounds such as ascorbic acid, L-glutamic acid, uric acid, creatine, glucose and dopamine have been investigated. The nature of the interaction between manganese metal cations and natural bilirubin is also discussed employing NPA, molecular orbital (MO) analysis and Bader’s Atoms in Molecule (AIM) theory.