Serum albumin (HSA) is the most abundant carrier protein present in the circulatory system. The drug-serum albumin interaction plays a dominant role in the metabolism of drugs, its distribution, free concentration, and efficacy. Also, numerous exogenous and endogenous compounds bind to serum albumin as well alkaloids and flavonoids. Flavonoids belong to a group of phenolic compounds found in fruits and vegetables. Olanzapine (OLZ) is an atypical antipsychotic drug is widely used for the treatment of schizophrenia, bipolar disorder, and related psychiatric disorders. The aim of this study was to investigate the binding properties of OLZ to HSA and its competitive binding to HSA with flavonoids - diosmin (DIO), quercetin (QUE) and catechin (CAT) under the physiological conditions by fluorescence and absorption spectroscopy. Results of fluorescence experiments suggest that OLZ quench the fluorescence of HSA through the mixed quenching mechanism and non-radiation energy transferring because of the HSA–OLZ complex formation. OLZ spontaneously bind in the site I on HSA, and according to thermodynamic parameters, the reaction was spontaneous and mainly driven by hydrogen bonds and van der Waals interactions. The presence of DIO and CAT decreased binding affinity between OLZ and HSA which indicates that they could compete against OLZ in the site I. Contrary, in the presence of QUE the binding affinity of the HSA-OLZ system enhanced, which may be explained by conformational changes in HSA (non-competitive interference). This study may provide a better understanding of OLZ pharmacokinetics and illustrate drug-drug interactions.