The effects of nitrate on the spectral characteristics and complexation ability of DOM with Cu²⁺ in the secondary effluent of municipal wastewater were investigated by using three-dimensional fluorescence and UV spectrophotometry. The fluorescence peak representing the protein-like (tryptophan-like) produced by complexation of DOM and Cu²⁺ disappeared with the increase of NO₃^- concentration, and the right side of the UV absorption peak occurred red-shift. The concentration of NO₃^- was significantly positively correlated with the values of HIX, α₃₀₀, α₂₅₀/α₃₆₅, E₃/E₄ and UV₂₅₃/UV₂₀₃ after complexation of DOM with Cu²⁺, while negatively correlated with BIX (r with -0.958~-0.719). It had an exponential increase with S_240-400 (R² with 0.9678~0.9952) and an exponential decrease with S_275-295 (R² with 0.9965~0.9978), but had little effect on the values of FI, α₃₅₀, α₃₅₅ and S_350-400. The conditional stability constant lgK of ultraviolet humus-like, visible humus-like and high excitation wavelength tryptophan-like in the secondary effluent of DOM with Cu²⁺ were 2.08~2.46L/mmol, 1.84~2.51L/mmol and 2.27~2.57L/mmol, respectively; and the proportion of coordination fluorescent functional groups were 19.5%~36.5%, 22.5%~33.3% and 24.4%~29.2%, respectively. In summary, there was not significant difference in the intensity of the complexation reaction between Cu²⁺ and humus-like or protein-like from DOM of the secondary effluent. When the NO₃^- concentration in the samples was different, there were some uncertainties in expressing the content of DOM and molecular properties with partial fluorescence (HIX) and ultraviolet parameters (α₃₀₀, α₂₅₀/α₃₆₅, E₃/E₄, S_240-400 and S_275-295).