The study aimed to conduct chemical profiling, acute in-vivo toxicity evaluation, and the potential anti-diabetic effect of standardized Aloe sabaea flowers ethanolic extracts (ASFEE) on alloxan-induced diabetic rats. The chemical composition was analyzed using GC–MS and TLC techniques. The oral acute toxicity study was performed according to the WHO 2000 and the OECD 420 guidelines. Furthermore, anti-diabetic activity was investigated using two doses of ASFEE (0.2 and 0.5 g/kg/day BW, p.o.) compared with glibenclamide (5 mg/kg/day, p.o.). A molecular docking investigation of the identified components with the PTPN9 enzyme was performed to figure out the proposed anti-diabetic mechanism. GC–MS analysis displayed the existence of 18 compounds; most of the compounds were fatty acids and their esters, and phytosterols. Total phenolic and flavonoid contents were 42.00±1.26 mg GAE/g DW and 22.21±1.55 mg QE/g DW, respectively. The results of the in-vivo toxicity study revealed the absence of noticeable signs of toxicity or mortality at various doses establishing the safety of the tested extract. The estimated LD50 value was higher than 10 g/kg. Antidiabetic action exhibited a noticeable decline in fasting blood glucose (FBG) levels comparable to glibenclamide with no inducing intense hypoglycemia and considerable excess weight.