Herein, a 3-channel band-bass filter (BPF) turntable was proposed to perform electrochemiluminescence (ECL) multiplexing immunoassay (MIA) in the spectrum- and potential-resolved determinations in a normal ECL analyzer. The simultaneous quantification of α-fetoprotein (AFP), carbohydrate antigen 125 (CA125) and carbohydrate antigen 19–9 (CA19-9) was achieved in one cathodic potential scan run. Stopped the BPF turntable at the indicator channel, AFP was determined in the potential range from 0 to –0.8 V by using perylene tetracarboxylic acid@ carboxylic carbon nanotubes (Ep = –0.43 ∼ –0.45 V) as the ECL tag, with the limit of detection (LOD) of 0.53 fg mL−1. During the potential scanned from –0.8 to –1.6 V then back –0.8 V, the BPF turntable was rotated to perform the spectrum-resolved ECL MIA for CA125 and CA19-9, with nanocrystals of CdSe (λmax = 550 nm) and CdTe (λmax = 750 nm) as the ECL tags, respectively. A simple accumulative algorithm was used to obtain stable ECL intensity for spectrum measurement and enhance the sensitivity in ECL MIA. The LODs for CA125 and CA19-9 were 0.058 and 0.11 mU mL−1, respectively, which are close to those in the single immunoassays. The combination of the specially designed BPF turntable and single photomultiplier tube detector offers high sensitivity in the spectrum-resolved ECL biosensors.