Proteins are excellent templates and stabilizers for Au nanoclusters (NCs) because of their abundant thiol groups and unique internal environments. However, high-molecular weight (MW) proteins with special quaternary structures are rarely reported as such templates. Considering that proteins may afford different spatial configurations as templates for Au NCs, we focused on alkaline phosphatase, catalase, and fibrinogen (MW range from 150 to 340 kDa) as direct templates for synthesizing Au NCs. We demonstrated that both Cu²⁺ and Hg²⁺ could induce photoluminescence (PL) quenching of these Au NCs, while their binding mechanisms were different. Therefore, significant PL recovery by amino acids, e.g., histidine and cysteine, was observed for Cu²⁺-treated Au NCs, but not Hg²⁺-treated Au NCs, allowing for selective detection of Hg²⁺ by using histidine as a masking agent. The detection ranges were 0.06–2.0 μM for Hg²⁺ and 0.04–5.0 μM for Cu²⁺, with low limits of detection of 0.02 and 0.01 μM, respectively. The PL change showed opposite tendency for histidine and cysteine at higher concentrations, resulting in different PL outputs. Using dual metal ion and dual amino acid combinations, an integrated PL logic gate was fabricated. This work improves the understanding of the PL mechanisms of complicated protein-localized Au NCs.