Plants produce various specialized metabolites that have evolved in different species due to adaptation to specific ecological conditions. Flavonoid dimers or biflavonoids consist of two identical or different flavonoid monomers. These compounds are less researched than the monomers and have different biological activities, although their pharmacological potential has recently been increasingly recognized. In order for biflavonoids to be used as antibacterial, anti-inflammatory, antiallergic, antimutagenic, antiviral or anticarcinogenic agents, it is necessary to isolate them from natural sources. In this study, different methods for the extraction of biflavonoids from ginkgo leaves were investigated, including conventional extraction in 70% ethanol, ultrasound-assisted (UAE + 70% ethanol), mechanically assisted (MAE + 70% ethanol), chemically assisted (CAE + 70% ethanol) and enzyme-assisted extraction (EAE + 70% ethanol) using a mixture of lignocellulolytic enzymes (cellulase, laccase, xylanase). Morphological analysis of the powder before and after extraction was performed using a scanning electron microscope (SEM). The total content of polyphenols, phenolic acids and flavonoids in the extracts was determined spectrophotometrically, while the biflavonoids were identified and quantified by HPLC-DAD analysis. The study showed significant differences in extraction efficiency within the first 5 minutes between the different methods. Assisted extraction techniques significantly increased the efficiency of biflavonoid extraction, with enzyme-assisted extraction yielding a 50% increase in biflavonoid concentration compared to extraction with 70% ethanol alone. The most effective results were obtained with the UAE + 70% ethanol and EAE + 70% ethanol methods when the extraction was performed for 45 minutes.