Cellulose nanospheres (CN) have been considered a leading type of nanomaterial that can be applied as a strengthening material in the production of nanocomposites. This work aimed to isolate and characterize the properties of CN from different agricultural by-products. CNs were successfully isolated from rice straw, corncob, Phulae pineapple leaf and peel using acid hydrolysis (60% H2SO4) combined with homogenization-sonication (homogenized at 12,000 rpm for 6 min and ultrasonicated for 10 min). The results showed that the CN from rice straw (RS-CN) and corncob (CC-CN) exhibited high yields (22.27 and 22.36%) (p < 0.05). All hydrolyzed CNs exhibited a spherical shape with a diameter range of 2 to 127 nm. After acid hydrolysis, Fourier transform infrared (FTIR) results showed no impurities. X-ray diffraction (XRD) showed that the structure of cellulose was changed from cellulose-I to cellulose-II. However, cellulose-I remained in pineapple peel cellulose nanosphere (PP-CN). The crystalline index (CI) ranged from 43.98 to 73.58%, with the highest CI obtained in the CC-CN. The CN from all sources presented excellent thermal stability (above 300 °C). The functional properties, including water absorption Index (WAI), water solubility index (WSI) and swelling capacity were investigated. PP-CN showed the highest WAI and swelling capacity, while the PL-CN had the highest WSI (p < 0.05). Among all samples, CC-CN showed the highest extraction yield, small particle size, high CI, and desirable functional properties to be used as a material for bio-nanocomposites film.