In this study, four different assessment methods, Toxic Unit (TU), Additive Index (AI), Mixture Toxicity Index (MTI) and Similarity Parameter (λ), were used to evaluate the joint toxic effect of heavy metal hybrid systems (Cu2+, Cd2+, Zn2+, Pb2+, Cr3+, Co2+, Ni2+ and Sr2+) on photobacterium phosphoreum in three groups—EC30 mixture (Mix-R1), EC50 mixture (Mix-R2) and the equimolar ratio mixture (Mix-R3)—planned by the fixed ratio method. An orthogonal experiment was designed to evaluate and screen out the heavy metals that have significant influence on joint toxicity of the hybrid system. The results indicated that joint toxicities of the EC30 mixed-system (Mix-R1) and the EC50 mixed-system (Mix-R2) were analogous and differed from the equimolar-mixed system (Mix-R3), and joint toxic effects were not consistent in the same mixed system by the four evaluation methods. The joint toxicity of both the EC30 mixed system and the EC50 mixed system evaluated by the four evaluation methods (TU, AI, MTI and Similarity Parameter-λ) exhibited partly additive effect and antagonism effect and partly additive effect and antagonism effect separately. In the equimolar mixed system, only the antagonism effect was shown. The orthogonal experiment demonstrated that the joint toxic effect of the hybrid system was not dependent on a single metal. This study confirmed that varying the ratios of the mixture has an impact on the results of the evaluation.