Background and Purpose The development of biased agonism provides a promising avenue to improve the pharmacological properties of fentanyl derivatives, but the molecular mechanism underlying ligand bias still remains ambiguous. Therefore, we sought to find out the critical sites of μ-receptor governing ligand bias and clarify corresponding molecular mechanism for designing and synthesizing effective analgesics with reduced adverse effects. Experimental Approach Critical sites governing ligand bias were identified both by computational prediction and cell assay-based bias analysis on wild-type and site-directed mutant μ-opioid receptor. Then molecular dynamics simulations of wild-type and mutant μ-opioid receptor were conducted to investigate the mechanism of bias activation. Key Results D3.32A and H6.52L mutation disrupted the binding of fentanyl derivatives with μ-opioid receptor. W6.48L mutation drove most fentanyl derivatives to β-arrestin-bias but promote sufentanil to cAMP signaling-bias. The result of molecular dynamics simulation showed that W6.48 and Y7.43 were paired activation switches of ligand bias at μ-opioid receptor. Conclusion and Implications D3.32 and H6.52 were critical residues in driving morphine and fentanyl derivatives to bind with μ-opioid receptor. W6.48 was a pivotal residue in governing the bias signaling and the interactions of ligands with W6.48 and Y7.43 were the structural determinants for the signaling bias of μ-opioid receptor, which will be conducive for better design and synthesis of effective opioid analgesics with the reduced adverse effects.