Cassava leaves are rich in abundant flavonols, and efficient extraction and analysis methods are crucial for the evaluation of flavonol content in the cassava leaves. This study aims to optimize the extraction and detection methods for four flavonols (myricetin, rutin, nicotiflorin, narcissoside) in cassava leaves and to analyze the influence of different cassava varieties, harvesting periods, and maturity on the content of these flavonols. The results indicated that using a 50% ethanol-water solution, a liquid-to-material ratio of 1∶50 (g/mL), an ultrasonic extraction temperature of 50 ℃, and an ultrasonic extraction time of 60 minutes could effectively extract the four flavonols in the cassava leaves. The combination of different C18 chromatographic columns in HPLC-DAD effectively separated the four flavonoids present in the cassava leaves. The results of the method validation showed that the four flavonols exhibited good linear correlations within a certain concentration range, with R² values of 0.9999, 0.9999, 0.9999 and 0.9998, respectively. The detection limits ranged from 6.0 mg/kg to 10.0 mg/kg, and the quantification limits ranged from 20.0 mg/kg to 32.0 mg/kg. The detection method demonstrates good system adaptability, with retention time and peak area relative standard deviations (RSD) being less than 1%. The samples showed good stability in terms of intra-day, inter-day, and intra-month variations, with RSD in content variations ranging from 0.44% to 3.57%. The average recovery rates of the method ranged from 92.68% to 109.14%, all with RSD values less than 6.0%. Utilizing the established extraction and analysis methods, the study analyzed the content of the four flavonols in 30 cassava germplasm resources. Rutin and nicotiflorin maked up 93.50% to 99.30% of the total flavonol content in the cassava leaves, with the levels primarily determining the total flavonol content. However, the correlation order for the total flavonol content among different varieties was rutin>narcissoside>nicotiflorin>myricetin. Analysis of flavonol content in the cassava leaves from different harvest times and maturity levels of cassava germplasm revealed that in most cases, the levels of rutin, nicotiflorin and narcissoside were higher in the leaves harvested at 270 days compared to those harvested at 180 days, with myricetin varying by variety. Except for SC09, the levels of myricetin, rutin and nicotiflorin were higher in the young leaves compared to the tender and mature leaves in different cassava germplasms. The content of narcissoside in mature leaves (except for flower leaf cassava) was higher than that in young leaves and tender leaves. The results would provide an evaluation basis for the development and utilization of cassava flavonols in the selection of raw materials and quality control, and lay a foundation for revealing the accumulation rules of cassava flavonols.