Chlortetracycline (CTC) usually presents in livestock wastewater with oxytetracycline (OTC), causing damage to human health and ecosystems. It’s urgent to seek low-cost and ecofriendly technology for antibiotics removal. In this study, effects of CTC and CTC + OTC (CTC:OTC= 1:1, g/g) on Spirulina platensis have been investigated. EC50 value of CTC for S. platensis was 8.76 mg/L at 96 h and risk quotient value in wastewater was 15.85. Inhibition of CTC on S. platensis gradually enhanced with increase of CTC, but CTC + OTC below 1.0 mg/L didn’t harm the growth of microalgae. Chlorophyll-a (Chl-a) and carotenoid content showed a parabolic trend with extension of time at CTC group. Chl-a synthesis gradually decreased with increase of CTC + OTC stress. High concentrations of CTC and CTC + OTC showed obvious inhibition on phycocyanin production. Polyunsaturated fatty acids (PUFA) and saturated fatty acids (SFA) contents peaked at 1.0 mg/L CTC, corresponding to the minimum of superoxide dismutase (SOD) activity in S. platensis. SFA and PUFA contents decreased when CTC + OTC content was above 2.0 mg/L. CTC and CTC + OTC (both over 2.0 mg/L) stimulated production of dissolved extracellular organic matters in S. platensis. Removal efficiency of CTC by S. platensis was about 98.63–99.95% and its biodegradation pathways were hydroxylation and side-chain breakdown. Display omitted •Half maximal effective concentration for Spirulina platensis was 8.76 mg/L at 96 h.•Risk quotient value indicated CTC had high risk to S. platensis in wastewater.•Inhibition rate of CTC on S. platensis was more than that of CTC + oxytetracycline.•High CTC improved yield of dissolved extracellular organic matters in S. platensis.•Hydroxylation and side-chain breakdown were the main biodegradation pathways of CTC.