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Abstract Thioredoxin reductase （TrxR, enzyme code [E.C.] 1.6.4.5） is a widely distributed flavoenzyme that catalyzes nicotinamide adenine dinucleotide phosphate （NADPH）‐dependent reduction of thioredoxin and many other physiologically important substrates. Spirulina platensis is a blue–green algae that is often used as a dietary supplement. S. platensis is rich in protein, lipid, polysaccharide, pigment, carotenoid, enzyme, vitamins and many other chemicals and exhibits a variety of pharmacological functions. In the present study, a simple and efficient method to purify TrxR from S. platensis tablets is reported. The extractions were carried out using two different methods: heat denaturation and 2′,5′‐adenosine diphosphate Sepharose 4B affinity chromatography. The enzyme was purified by 415.04‐fold over the crude extract, with a 19% yield, and specific activity of 0.7640 U/mg protein. Optimum pH, temperature and ionic strength of the enzyme activity, as well as the Michaelis constant （ K m） and maximum velocity of enzyme （ V max） values for NADPH and 5,5′‐dithiobis（2‐nitrobenzoic acid） were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag+, Cu2+, Mg2+, Ni2+, Pb2+, Zn2+, Al3+, Cr3+, Fe3+, and V4+ ions; vitamin B3, vitamin B6, vitamin C, and vitamin U and aciclovir, azithromycin, benzyladenine, ceftriaxone sodium, clarithromycin, diclofenac, gibberellic acid, glurenorm, indole‐3‐butyric acid, ketorolac, metformin, mupirocin, mupirocin calcium, paracetamol, and tenofovir had inhibitory effects on TrxR. Ag+ exhibited stronger inhibition than 1‐chloro‐2,4‐dinitrobenzene （a positive control）.
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