To ensure food safety from animal origins and to prevent potential hazards caused by steroid hormones, there is an urgent need to develop a rapid detection method for steroid hormones in foods from animal origins. Three highly sensitive monoclonal antibodies (mAb) against testosterone propionate (Tp), stazolol (STA) and estradiol benzoate (EB) were prepared, with IC50 values of 0.13, 0.14 and 0.19 ng/mL, respectively. Tp-mAb is a group-selective mAb that simultaneously detects Tp, testosterone (T), methyltestosterone (MT) and testosterone phenylpropionate (TPP), and their IC50 values were 0.13, 0.77, 0.90, and 1.25 ng/mL, respectively. A novel multiplex lateral flow immunochromatographic strip (ICS) was further prepared, which could simultaneously detect Tp, STA, and EB in chicken and pork samples with limits of quantification (LOQ) of 0.32, 0.51, 0.35 μg/kg and 0.36, 0.64, 0.43 μg/kg, respectively. Based on the broad spectrum of Tp-mAb, ICS has been further developed to detect Tp, T, MT and TPP simultaneously. This multiplex ICS rapid detection method was efficient, sensitive, and fast, for the simultaneous detection of multiple steroid hormones in actual samples. Its detection time is only 40 min, which it represents a favorable monitoring tool for the detection of steroid hormones in animal-derived foods from the marketplace.