High-throughput and low-cost detection of mycotoxins in complex matrices is becoming increasingly urgent but it is still challenging to perform ultrasensitive analyses. Here we report a green and practical cytometric microbead magnetic suspension array (CBMSA) strategy for rapid and economical detection of aflatoxin B1 (AFB1) in multiple batches of lotus seed samples. The protocol included (1) fabrication of suspension array chips by immobilizing biotin-modified bovine serum albumin-AFB1 (antigen) onto the surface of streptavidin-coated magnetic microbeads in a multiwell array, (2) indirect immunocompetition of antigen and target of AFB1 in lotus seed samples with the specific antibodies, (3) rapid magnetic separation regardless of complex pretreatment steps, and (4) ultrasensitive fluorescence detection of fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin G (FITC-IgG) probes. After systematic optimization of some crucial parameters, the developed CBMSA assay allowed for ultrasensitive detection of AFB1 with limit of detection as low as 7.8125 pg·kg-1. For high-throughput analysis, the CBMSA technique was capable of on-site co-instantaneous detection of 50-100 samples in one operation within 30 s, only needing a small amount (50 μL) of solution, which is much cheaper, greener, and more user-friendly than conventional techniques. Moreover, CBMSA with magnetic separation is free of multiple centrifugation and cleanup steps to avoid unpredictable loss of targets. Since various capture and fluorescent probes can be randomly constructed and bound onto the surface of magnetic microbeads to establish an ultrasensitive detection system, the CBMSA technique is very promising for more trace analytes in complex matrices and for broad point-of-need applications, such as drug screening and real-time high-throughput analysis.