Nanocrystalline spinel ferrite nanoparticles MxCo(1−x)Fe2O4;(M = Zn,Cu,Mn;x = 0 and 0.5) like: Cobalt ferrite (CFO), Zinc Cobalt ferrite (ZCFO), Copper Cobalt ferrite (CCFO), and Manganese Cobalt ferrite (MCFO) modified carbon paste electrodes (CPE) were synthesized via sol–gel technique utilizing citric acid and ethylene glycol as a polymerization agent. The synthesized ferrite NPs were used as bi-functional smart biosensor, not only used to determine the drug Anagrelide-HCl (ANDH) in urine and serum samples, but also possesses antimicrobial potential against some pathogenic microbes, founded in the biological samples. The synthesized ferrite NPs were confirmed by XRD, FTIR spectroscopy, SEM, EDX, and elemental mapping images. Antimicrobial activities of ferrite NPs against selected urinary tract infected microbes were investigated. From XRD data and FTIR spectroscopy it is found that the average crystallite size is lies in the range 12.86 to 33.92 ± 1.5 nm, also the bond lengths RA and RB increase from 1.8986 to 1.9145 Å and from 2.0434 to 2.0606 Å respectively and Debye temperature θD lies in the range of 681.52–708.87 K. Our study describes the improvement of a screen-printed sensor, modified with ferrite NPs materials for rapid, sensitive and cost-effective quantification of ANDH present in the real samples such as blood serum samples, urine and in the pharmaceutical formulations. The results obtained postulate a linear regression between the ANDH charge density of peak current and its concentration in the range from (0.64–8.18 μg/ml) with DL 0.31 μg/ml and QL 0.94 μg/ml. Antimicrobial results indicated that ZCFO NPs were a novel antibacterial agent against Klebsiella pneumoniae (28.0 mm ZOI), and multidrug-resistant bacteria Enterococcus faecalis (27.0 mm ZOI). Additionally, ZCFO NPs were active against Candida albicans (18.0 mm ZOI) seems to be a smart antifungal agent. Therefore, ZCFO NPs can be used as applicant resources for industrial, medical, and biological applications. Display omitted •Synthesis and validation of spinel ferrite NPs using citrate sol-gel technique.•The theoretical values of lattice constant for ferrite NPs are approximated to experimentally lattice parameter values.•The Debye temperature for the ferrite NPs composition lies in the range of 681.52 – 708.87 K.•ZCFO NPs possessed a novel antimicrobial activity against selected MDR pathogens.•Improvement of a novel ZCFO NPs screen-printed sensors for rapid, sensitive and cost-effective ANDH quantification.