Existing research methods for biological effects of extremely low frequency electromagnetic fields (ELF-EMFs) are comparison between groups, with inability to exclude individual differences in cell sensitivity or changes in conditions during experiment. This work proposed a method for real-time effects observation of ELF-EMFs in the same cell and same condition. The stability domain was used to identify the stability of hippocampal neurons before ELF-EMFs exposure. At the time of electromagnetic field intervention (t=60 s), ELF-EMFs were exposed to hippocampal neurons at 0, 0.09, 0.38, 0.76, 7.33 and 14.78 mT, respectively. Reactive oxygen species (ROS) and Ca²⁺ fluorescence response curves of hippocampal neurons were recorded in real-time, and the autocorrelation function between ELF-EMFs and fluorescence response was established. The results showed as follows: (1) The step properties of fluorescence response were clear at the burst times of ROS and Ca²⁺, which were important indicator to identify the real-time response; (2) The response time of ROS and Ca²⁺ to ELF-EMFs were delayed and inconsistent; (3) ROS and Ca²⁺ had dose-dependent response to ELF-EMFs; (4) The response of ROS to ELF-EMFs was complex; (5) The response of Ca²⁺ to ELF-EMFs was asymptotically stable. When correlation function exceeded 0.3, the real-time response of ELF-EMFs and ROS/Ca²⁺ was correlated. It was concluded that a real-time response method of intracellular ROS and Ca²⁺ to ELF-EMFs exposure was feasible for the evaluation of electromagnetic bioeffects.