Toxicity interaction, synergism and antagonism, may occur when multiple pollutants are exposed to the environment simultaneously, which limits the utility of some standard models to assess toxicity hazards and risks. The development and application of models which can provide an insight into the combined toxicity of pollutants becomes necessary. Therefore, a novel model, area-concentration ratio (ACR) method, was developed to characterize the toxicity interaction within mixtures of three aminoglycoside antibiotics (AGs), kanamycin sulfate (KAN), paromomycin sulfate (PAR), tobramycin (TOB) and one heavy metal copper (Cu) in this study. The inhibition toxicity of single contaminants and mixtures designed by direct equilibration ray method and uniform design ray method to Chlorella pyrenoidosa (C. pyrenoidosa) was determined by the microplate toxicity analysis (MTA). The results showed that the novel method ACR could be used for quantitative characterization of combined toxicity. According to the ACR, all the binary AG antibiotic mixture systems display obvious synergism and weak antagonism. The addition of the heavy metal Cu into binary AG antibiotic mixtures can obviously change toxicity interaction, but toxicity interaction changing trend varies greatly in different ternary mixture systems. Toxicity interaction in the six mixture systems has component concentration-ratio dependence. ACR can be suggested as an effective novel method to quantitatively characterize toxicity interaction when assessing the hazards and risks of the combined pollution.