In petrochemical industry, zeolites as solid acid catalysts play an important role and Y zeolites with high surface area and large pore openings are mainly used for catalytic cracking. Aiming at the problem that the specific positions of Al atoms in zeolites cannot be accurately distinguished experimentally and the acid strength can only be studied qualitatively, we used computational model and method to study the position of Al substitution and the resulted acid strength of Y zeolite at atomic and molecular level. The position of Al substitution with gradually decreasing Si/Al ratio (Si/Al = 47–2.4) and the corresponding Brønsted acid strength of the resulting multiple acid sites of HY zeolites are systematically investigated based on periodic DFT calculation including dispersion correction (GGA-PBE-D3). The thermodynamically more stable structures prefer Al distribution having the next-nearest neighbor of aluminum atom pairs (Al–O–Si–O–Al), and based on these stable configurations, the adsorption of ammonia (NH3) and pyridine (C5H5N) has been computed to estimate the Brønsted acid strength. It is found that the adsorption of NH3 and C5H5N prefers the formation of ammonium (NH4+) and pyridinium (C5H5NH+) ions at each acid sites and the adsorption enthalpy does not correlate with the Bader charge of the acid proton. Most importantly, the adsorption enthalpy decreases with the decrease of Si/Al ratio, indicating that the more the Al substitution the weaker the Brønsted acid strength. It is noted that the adsorption enthalpy of all individual acid sites at a given Si/Al ratio can differ strongly, despite the formation of NH4+ and C5H5NH + ions, indicating their mutual interaction and local confinement effect. The calculated adsorption enthalpies agree fully with the experimentally measured results (Si/Al = 8.6–2.4). Hyroxyl nest, defect sites formed from dealumination, has much weaker acid strength than the Brønsted acid sites. Our results provide a rational basis for further studies on the enhanced catalytic activity of HY zeolites with different Si/Al ratios.