Biogenic volatile organic compounds (BVOC) substantially impact regional photochemical air pollution, global climate change, and the carbon cycle. Although research on BVOC emissions is of paramount importance, only few studies have measured long-term BVOC fluxes from forest ecosystems. There are no long-term observational studies on BVOC emissions from suburban forests of major cities in Asia under a humid subtropical climate, which may have a major impact on urban air quality. We conducted long-term, multi-height BVOC observations at a 30 m flux tower in suburban Tokyo, evaluating isoprene emission flux from a <em>Quercus serrata</em> dominated mixed forest using the aerodynamic gradient method. Spatial variability was examined through integrated drone and tower observations, and Model of Emission Gases and Aerosols from Nature (MEGAN) estimates were compared with measurements. Isoprene volume mixing ratios increased significantly during the warm season (May–October), accounting for over 90% of BVOC composition in peak summer, while monoterpenes remained low with minimal vertical gradients. Isoprene exhibited distinct vertical volume mixing ratio gradients peaking within the canopy, with daily average emission flux ranging from −0.05 to 15.30 mg·m⁻²·h⁻¹. Horizontal volume mixing ratio variability within 30 m reached 10–30%, with enhanced heterogeneity in summer. Horizontal flux differed by approximately 30% between tower (height: 23–30 m) and drone (30–40 m) measurements. MEGAN systematically overestimated observations with maximum deviations in summer. These findings, derived from long-term observations, will contribute to assessing the impact of BVOCs on air quality and climate in cities worldwide, beyond temperate humid regions of Asia, and to reducing model uncertainties.