Seaweed can bioaccumulate nanomaterials that would be transferred to the trophic chain. This work describes the optimization of a method for the separation of silver nanoparticles (AgNPs) from seaweed using an ultrasound-assisted enzymatic hydrolysis method and ulterior determination by single particle inductively coupled plasma mass spectrometry (SP-ICP-MS). The following parameters affecting the isolation of AgNPs were optimized using a Palmaria palmata (red seaweed) sample previously exposed to AgNPs: type of sonication (bath vs. ultrasonic probe), ultrasound amplitude, sonication time, sonication mode (pulsed vs. continuous sonication), concentration of the enzymes mixture (Macerozyme R-10®), and enzymatic hydrolysis time. The stability of AgNPs during extraction was tested by transmission electron microscopy (TEM) and using a standard of 15 nm of polyvinylpyrrolidone (PVP)-coated AgNPs analyzed by SP-ICP-MS. The analytical performance was evaluated with good results. For total Ag determination, the limits of detection and quantification were 2.2 and 7.7 ng g−1, respectively; and for AgNPs determination, the limits of detection in size and number were 14 nm and 4.34 × 107 part g−1, respectively. Besides, the matrix effect, the repeatability and the analytical recovery were also studied. Finally, the method was applied to the analysis of several red (Palmaria palmata) and green (Ulva sp.) seaweed samples.